U.S. patent application number 13/437846 was filed with the patent office on 2013-01-03 for photon reducing agents for fluorescence assays.
This patent application is currently assigned to Life Technologies Corporation. Invention is credited to Tom KNAPP, Paul Negulesou, Timothy James Rink, Roger Y. Tsien, Gregor Zlokarnik.
Application Number | 20130004960 13/437846 |
Document ID | / |
Family ID | 26733118 |
Filed Date | 2013-01-03 |
United States Patent
Application |
20130004960 |
Kind Code |
A1 |
KNAPP; Tom ; et al. |
January 3, 2013 |
PHOTON REDUCING AGENTS FOR FLUORESCENCE ASSAYS
Abstract
The present invention provides a method for reducing undesirable
light emission from a sample using at least one photon producing
agent and at least one photon reducing agent (e.g. dye-based photon
reducing agents). The present invention further provides a method
for reducing undesirable light emission from a sample (e.g., a
biochemical or cellular sample) with at least one photon producing
agent and at least one collisional quencher. The present invention
also provides a method for reducing undesirable light emission from
a sample (e.g., a biochemical or cellular sample) with at least one
photon producing agent and at least one quencher, such as an
electronic quencher. The present invention also provides a system
and method of screening test chemicals in fluorescent assays using
photon reducing agents. The present invention also provides
compositions, pharmaceutical compositions, and kits for practicing
these methods.
Inventors: |
KNAPP; Tom; (Carlsbad,
CA) ; Zlokarnik; Gregor; (La Jolla, CA) ;
Negulesou; Paul; (Solana Beach, CA) ; Tsien; Roger
Y.; (La Jolla, CA) ; Rink; Timothy James;
(Labande, MC) |
Assignee: |
Life Technologies
Corporation
Carlsbad
CA
|
Family ID: |
26733118 |
Appl. No.: |
13/437846 |
Filed: |
April 2, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12861791 |
Aug 23, 2010 |
8163562 |
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13437846 |
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12398744 |
Mar 5, 2009 |
7785536 |
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12861791 |
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11047074 |
Jan 31, 2005 |
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12398744 |
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09759629 |
Jan 12, 2001 |
7067324 |
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11047074 |
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09122477 |
Jul 23, 1998 |
6221612 |
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09759629 |
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60054519 |
Aug 1, 1997 |
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Current U.S.
Class: |
435/6.13 ;
435/29; 435/8 |
Current CPC
Class: |
C12Q 1/682 20130101;
G01N 33/582 20130101; C12Q 1/682 20130101; Y10S 436/80 20130101;
Y10T 436/13 20150115; C12Q 2527/125 20130101; C12Q 2563/107
20130101; Y10S 436/809 20130101; G01N 33/542 20130101; G01N 33/5005
20130101 |
Class at
Publication: |
435/6.13 ;
435/29; 435/8 |
International
Class: |
G01N 21/64 20060101
G01N021/64; G01N 21/75 20060101 G01N021/75 |
Claims
1. A method of reducing undesired light emission from a sample,
comprising: contacting a sample in need of reducing undesired light
with an aqueous solution that comprises at least one photon
reducing agent, wherein said sample comprises a living cell,
wherein said cell comprises at least one photon producing agent and
wherein said at least one photon reducing agent is substantially
membrane impermeant, and detecting an optical signal from said at
least one photon producing agent.
2. The method of claim 1, wherein said at least one photon reducing
agent has an absorption spectra that overlaps with the absorption,
emission or excitation spectrum of said at least one photon
producing agent.
3. The method of claim 2, wherein said at least one photon
producing agent exhibits fluorescence resonance energy transfer
with said at least one photon reducing agent.
4-5. (canceled)
6. The method of claim 1, wherein said sample comprises at least
two photon reducing agents.
7. The method of claim 6, wherein at least one of said at least two
photon reducing agents are dyes.
8. (canceled)
9. The method of claim 1, wherein said at least one living cell is
a mammalian cell.
10. The method of claim 1, wherein said at least one photon
reducing agent is selected from the group consisting of a
collisional quencher, a particulate, an absorption quencher, a FRET
quencher and a dark complex.
11. The method of claim 1, wherein said at least one photon
reducing agent is a dye.
12. The method of claim 1, wherein said at least one living cell is
part of a plurality of cells comprising at least two different
photon producing agents.
13. The method of claim 9, wherein said at least one photon
producing agent corresponds to an enzyme activity inside said
mammalian cell.
14-17. (canceled)
18. The method of claim 1, wherein said detecting comprises
detecting a fluorescence signal.
19. The method of claim 18, wherein said at least one photon
producing agent is produced from a precursor molecule that is a
substrate for an esterase.
20. The method of claim 19, wherein said at least one photon
producing agent detects the presence of an ion inside said membrane
compartment.
21. The method of claim 19, wherein said at least one photon
producing agent is a fluorescent protein.
22. (canceled)
23. The method of claim 19, wherein said at least one photon
producing agent detects voltage across a membrane of said membrane
compartment.
24-48. (canceled)
49. A composition of matter, comprising: a) a living cell, wherein
said cell comprises at least one photon producing agent, and b) an
aqueous solution with at least one photon reducing agent, wherein
said at least one photon reducing agent is substantially membrane
impermeant and does not specifically bind to said cell.
50-55. (canceled)
56. The composition of matter of claim 49, wherein said at least
one photon reducing agent is selected from the group consisting of
a collisional quencher, a particulate, an absorption quencher, a
FRET quencher and a dark complex.
57-61. (canceled)
62. The composition of matter of claim 49, wherein said at least
one photon reducing agent is a dye.
63. (canceled)
64. The composition of matter claim 49, wherein said composition
further includes a microplate and said living cell is a member of a
plurality of living cells in a well of said microplate.
65-70. (canceled)
71. A method of identifying a chemical with a biological activity,
comprising: a) contacting a sample with a test chemical, said
sample comprising a target, b) contacting said sample with an
aqueous solution that comprises at least one photon reducing agent,
wherein said sample comprises a living cell, wherein said cell
comprises at least one photon producing agent that directly or
indirectly monitors the activity of said target and said at least
one photon reducing agent is substantially membrane impermeant, and
c) detecting an optical signal from said at least one photon
producing agent, wherein said at least one photon reducing agent
has an absorption spectra that overlaps with the absorption,
emission or excitation spectrum of said at least one photon
producing agent or wherein said at least one photon producing agent
can exhibit fluorescence resonance energy transfer with said at
least one photon reducing agent.
72-79. (canceled)
Description
[0001] This application is a Continuation of Ser. No. 09/122,477,
filed Jul. 23, 1998, which claims the benefit of priority under 35
U.S.C. .sctn.119(e) to U.S. provisional application No. 60/054,519,
filed Aug. 1, 1997.
TECHNICAL FIELD
[0002] The present invention generally relates to methods and
compositions for reducing undesired light from an assay sample,
particularly fluorescence assays in living cells.
BACKGROUND
[0003] Cell-based assays are commonly used for drug discovery to
screen large numbers of test chemicals for potential therapeutic
activity. Typically, the cells contain a target, such as a protein.
Test-chemicals, such as candidate ligands for a target protein, are
screened for modulating activity of a target. Screening relies on a
detectable change in a property of a cell that faithfully reports
target activity in the presence of a test chemical. Many assays use
optical methods to detect such activities. Fluorescence detection
methods are particularly powerful tools in this regard, because
fluorescence detection methods can be sensitive. Many different
types of fluorescent probes are available for such assays,
including fluorescent probes that act as enzyme substrates, labels
for proteins and nucleic acids, indicators of intra-cellular ions,
and sensors of membrane voltage.
[0004] Despite the recent plethora in available fluorescent tools
for assays, fluorescence based assays can be plagued by
undesirable, and sometimes intolerable, levels of background
fluorescence. For example, solution fluorescence may increase the
background fluorescence of the assay sample. Solution fluorescence
can obscure a desired signal associated with a fluorescent probe.
Solution fluorescence can arise from many sources, including
fluorescent probe degradation, targets, cells, various solution
components, and test chemicals.
[0005] In cell-based assays recently developed by one of the
inventors of the present invention, solution fluorescence can give
rise to lower signal to noise ratios. These cell-based assays
utilize a membrane permeable substrate specific for beta-lactamase,
a bacterial enzyme that is not normally expressed in mammalian
cells. The substrate diffuses into the cell and is trapped inside
the cell by the action of intracellular esterases. If a cell
expresses a beta-lactamase reporter gene, the expressed enzyme will
cleave the substrate. Before cleavage the substrate fluoresces
green and after cleavage the substrate fluoresces blue. When such
assays are used for high-throughput screening, increasing the
signal to noise ratio can be advantageous because it increases the
sensitivity of the screening system and reliability of the data.
Solution fluorescence, however, often thwarts achieving
advantageous signal to noise ratios. Solution fluorescence from
test chemicals, substrate in the solution, and other solution
components that bath the cells contribute to background
fluorescence.
[0006] The present inventors recognized that membrane compartment
assays, such as cell-based assays, that use optical methods could
be improved by reducing unwanted light emitted from the solution
bathing the membrane compartments, particularly solution
fluorescence. The present inventors investigated different washing
and incubation methods in an attempt to increase dye loading and
retention while reducing solution fluorescence. Although the
inventors could reduce solution fluorescence, such manipulations
were cumbersome and time consuming.
[0007] Consequently, the present inventors developed compositions
and methods for reducing the emission undesired light from
solutions in membrane compartment assays that did not solely rely
on washing or incubation methods. Such compositions and methods are
much more applicable to high-throughput screening than improvements
to washing and incubation methods.
BRIEF DESCRIPTION OF THE FIGURES
[0008] FIG. 1A is a diagram of fluorescence emission from a
solution without a photon reducing agent.
[0009] FIG. 1B is a diagram of fluorescence emission from a
solution with a photon reducing agent.
[0010] FIG. 2 shows the ability of phenol red to reduce the
emission of fluorescence from a solution containing coumarin.
[0011] FIG. 3 shows the ability of phenol red to reduce the
emission of fluorescence from a solution containing coumarin the
presence and absence of methanol.
[0012] FIG. 4 shows the dependence of emission of fluorescence from
a solution on candidate photon reducing agent concentration.
[0013] FIG. 5 shows the dependence of emission of fluorescence from
a solution containing coumarin in the presence of various photon
reducing agents.
[0014] FIG. 6 shows the dependence of emission of fluorescence from
a solution containing fluorescein in the presence of various
colored photon reducing agents.
[0015] FIG. 7 shows the dependence of emission of fluorescence from
a solution containing rhodamine in the presence of various colored
photon reducing agents.
[0016] FIG. 8 shows residual CCF2 solution fluorescence as a
function of colored photon reducing agent concentration.
[0017] FIG. 9A, FIG. 9B, and FIG. 9C show the reduction of solution
fluorescence using non-dye based photon agents that electronically
interact with a photon producing agent.
[0018] FIG. 10A, FIG. 10B, and FIG. 10C show coumarin, fluorescence
is attenuated by phenol red at various pathlengths.
[0019] FIG. 11 shows that photon reducing agents reduce the
fluorescence emission in unwashed cells and yields signals
comparable to signals from washed cells.
[0020] FIG. 12 summarizes the results of photon reducing agent
toxicity testing.
[0021] FIG. 13 shows the ability of cells treated with photon
reducing agents to express beta-lactamase after stimulation with an
appropriate agonist.
[0022] FIG. 14A and FIG. 14B show that photon reducing agent sets
can reduce undesired fluorescence better than single dye-based
photon reducing agents.
SUMMARY
[0023] The present invention provides for a method of reducing
light emission, such as undesirable light, from a sample, such as a
solution. The method can be used with fluorescent assays that are
often hampered by solution fluorescence that interferes with
detecting a desired signal from the sample. Membrane compartment
based assays, such as cell-based assays, typically exhibit
undesirable background fluorescence from probes, test chemicals, or
other solution components that can interfere with desired signal
detection. To overcome these problems, the inventors devised a
method to reduce undesired light emitted from the sample by adding
a photon reducing agent to the sample. These methods can be used,
for example, to identify a chemical with a biological activity. The
present invention also includes a therapeutic composition
identified by such methods and a system to perform such methods and
to identify a chemical with a toxicological or bioavailability
activity.
[0024] The present invention also provides a composition of matter
comprising a membrane compartment that is in physical or optical
contact with a solid surface, such as a surface that can transmit
light, and an aqueous solution with at least one photon reducing
agent. The solid surface can be, for example, a well of a
multi-well platform, such as a microtiter plate. Optionally, the
membrane compartment need not be in contact with a solid surface.
In this aspect of the present invention, the membrane compartment
can be within a drop or droplet such as they are generated during
FACS procedures.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0025] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
Generally, the nomenclature used herein, and the laboratory
procedures in spectroscopy, drug discovery, cell culture, and
molecular genetics, described below, are those well known and
commonly employed in the art. Standard techniques are typically
used for signal detection, recombinant nucleic acid methods,
polynucleotide synthesis, and microbial culture and transformation
(e.g., electroporation, and lipofection). The techniques and
procedures are generally performed according to conventional
methods in the art and various general references (see generally,
Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed.
(1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y.; and Lakowicz, J. R. Principles of Fluorescence Spectroscopy,
New York: Plenum Press (1983)) for fluorescence techniques, each of
which are incorporated herein by reference) which are provided
throughout this document. Standard techniques are used for chemical
syntheses, chemical analyses, and biological assays. As employed
throughout the disclosure, the following terms, unless otherwise
indicated, shall be understood to have the following meanings:
[0026] "Fluorescent donor moiety" refers to the radical of a
fluorescent compound that can absorb energy and is capable of
transferring the energy to an acceptor, such as another fluorescent
compound or another part of the fluorescent compound. Suitable
donor fluorescent compounds include, but are not limited to,
coumarins and related dyes, xanthene dyes such as fluoresceins,
rhodols, and rhodamines, resorufins, cyanine dyes, bimanes,
acridines, isoindoles, dansyl dyes, aminophthalic hydrazides such
as luminol, and isoluminol derivatives, aminophthalimides,
aminonaphthalimides, aminobenzofurans, aminoquinolines,
dicyanohydroquinones, and fluorescent europium and terbium
complexes and related compounds.
[0027] "Quencher" refers to a molecule or part of a compound that
is capable of reducing the emission from a fluorescent moiety. Such
reduction includes reducing the light after the time when a photon
is normally emitted from a fluorescent moiety. Quenching may occur
by any of several mechanisms, including fluorescence resonance
energy transfer, photoinduced electron transfer, paramagnetic
enhancement of intersystem crossing, Dexter exchange coupling, and
excitation coupling, such as the formation of dark complexes.
[0028] "Acceptor" refers to a quencher that operates via energy
transfer. Acceptors may re-emit the transferred energy as
fluorescence. Examples of acceptors include coumarins and related
fluorophores, xanthenes such as fluoresceins, rhodols, and
rhodamines, resorufins, cyanines, difluoroboradiazaindacenes, and
phthalocyanines. Other chemical classes of acceptors generally do
not re-emit the transferred energy. Examples include indigos,
benzoquinones, anthraquinones, azo compounds, nitro compounds,
indoanilines, and di- and triphenylmethanes.
[0029] "Binding pair" refers to two moieties (e.g. chemical or
biochemical) that have an affinity for one another. Examples of
binding pairs include antigen/antibodies, lectin/avidin, target
polynucleotide/probe oligonucleotide, antibody/anti-antibody,
receptor/ligand, enzyme/ligand and the like. "One member of a
binding pair" refers to one moiety of the pair, such as an antigen
or ligand.
[0030] "Dye" refers to a molecule or part of a compound that
absorbs frequencies of light, including, but not limited to,
ultraviolet light. The terms "dye" and "chromophore" are
synonymous.
[0031] "Fluorophore" refers to a chromophore that fluoresces.
[0032] "Membrane-permeant derivative" refers to a chemical
derivative of a compound that has enhanced membrane permeability
compared to an underivativized compound. Examples include ester,
ether and carbamate derivatives. These derivatives are made better
able to cross cell membranes (i.e. membrane permeant) because
hydrophilic groups are masked to provide more hydrophobic
derivatives. Also, masking groups are designed to be cleaved from a
precursor (e.g., fluorogenic-substrate precursor) within a cell to
generate the derived substrate intracellularly. Because the
substrate is more hydrophilic than the membrane permeant
derivative, it becomes trapped within the cell. Membrane-permeant
and membrane-impermeant are relative terms based on the
permeability characteristics of a compound and a chemical
derivative thereof.
[0033] "Isolated polynucleotide" refers a polynucleotide of
genomic, cDNA, or synthetic origin, or some combination there of,
which by virtue of its origin the "isolated polynucleotide" (1) is
not associated with the cell in which the "isolated polynucleotide"
is found in nature, or (2) is operably linked to a polynucleotide
which it is not linked to in nature.
[0034] "Isolated protein" refers a protein, encoded by cDNA,
recombinant RNA, or synthetic nucleic acids, or some combination
thereof, which by virtue of its origin the "isolated protein" (1)
is not associated with proteins that it is normally found with in
nature, (2) is isolated from the cell in which it normally occurs,
(3) is isolated free of other proteins from the same cellular
source (e.g. free of human proteins), (4) is expressed by a cell
from a different species, or (5) does not occur in nature.
"Isolated naturally occurring protein" refers to a protein which by
virtue of its origin the "isolated naturally occurring protein" (1)
is not associated with proteins that it is normally found with in
nature, or (2) is isolated from the cell in which it normally
occurs, or (3) is isolated free of other proteins from the same
cellular source, e.g. free of human proteins.
[0035] "Polypeptide" as used herein as a generic term to refer to
native protein, fragments, or analogs of a polypeptide sequence.
Hence, native protein, fragments, and analogs are species of the
polypeptide genus.
[0036] "Naturally-occurring" as used herein, as applied to an
object, refers to the fact that an object can be found in nature.
For example, a polypeptide or polynucleotide sequence that is
present in an organism (including viruses) that can be isolated
from a source in nature and which has not been intentionally
modified by man in the laboratory is naturally-occurring.
[0037] "Operably linked" refers to a juxtaposition wherein the
components so described are in a relationship permitting them to
function in their intended manner. For example, a control sequence
"operably linked" to a coding sequence is linked (for example,
ligated) in such a way that expression of the coding sequence is
achieved under conditions compatible with the control sequences,
such as when the appropriate molecules (e.g., inducers and
polymerases) are bound to the control or regulatory
sequence(s).
[0038] "Control sequence" refers to polynucleotide sequences that
are necessary to effect the expression of coding and non-coding
sequences to which they are linked (for example, ligated). The
nature of such control sequences differs depending upon the host
organism. In eukaryotes, such control sequences generally include
enhancers, promoters, ribosomal binding sites, and transcription
termination sequences. In prokaryotes, generally, such control
sequences include promoters and transcription termination sequence.
The term "control sequences" is intended to include, at a minimum
components whose presence can influence the expression of a gene,
and can include additional components whose presence is
advantageous, for example, leader sequences and fusion partner
sequences (for example, sequences encoding a fusion protein).
[0039] "Polynucleotide" refers to a polymeric form of nucleotides
of at least 10 bases in length, either ribonucleotides or
deoxynucleotides or a modified form of either type of nucleotide.
The term includes single and double stranded forms of DNA.
[0040] "Corresponds to" refers to a sequence that is homologous
(i.e., is identical, not strictly evolutionarily related) to all or
a portion of a reference sequence.
[0041] "Membrane compartment" refers to a semi-permeable material
(for example, a biological membrane, vesicle, cell (for example,
prokaryotic or eukaryotic, such as mammalian, such as human),
liposome, envelope of a virus, or the like, surrounding a volume of
aqueous fluid, such as intracellular fluid.
[0042] "Modulation" refers to the capacity to either enhance or
inhibit a functional property of biological activity or process
(e.g., enzyme activity or receptor binding). Such enhancement or
inhibition may be contingent upon the occurrence of a specific
event, such as activation of a signal transduction pathway, and can
be exhibited only in particular cell types.
[0043] The term "modulator" refers to a chemical compound
(naturally occurring or non-naturally occurring), such as a
biological macromolecule (e.g., nucleic acid, protein, non-peptide,
or organic molecule), or an extract made from biological materials
such as bacteria, plants, fungi, or animal (particularly mammalian,
including human) cells or tissues. Modulators are evaluated for
potential activity as inhibitors or activators (directly or
indirectly) of a biological process or processes (e.g., agonist,
partial antagonist, partial agonist, inverse agonist, antagonist,
antineoplastic agents, cytotoxic agents, inhibitors of neoplastic
transformation or cell proliferation, cell proliferation-promoting
agents, and the like) by inclusion in screening assays described
herein. The activity of a modulator may be known, unknown or
partially known.
[0044] The term "test chemical" refers to a chemical to be tested
by one or more screening method(s) of the invention as a putative
modulator. A test chemical can be any chemical, such as an
inorganic chemical, an organic chemical, a protein, a peptide, a
carbohydrate, a lipid, or a combination thereof.
[0045] The terms "label" or "labeled" refers to incorporation of a
detectable marker. For example, by incorporation of a radiolabeled
amino acid or attachment to a polypeptide of biotinyl moieties that
can be detected by marked avidin (e.g., streptavidin containing a
fluorescent marker or enzymatic activity that can be detected by
optical or colorimetric methods). Various methods of labeling
polypeptides and glycoproteins are known in the art and may be
used. Examples of labels for polypeptides include, but are not
limited to, the following: radioisotopes (e.g., .sup.3H, .sup.14C,
.sup.35S, .sup.125I, .sup.131I), fluorescent labels (e.g., FITC,
rhodamine, lanthanide phosphors), enzymatic labels or a product of
a reporter gene (e.g., horseradish peroxidase, beta-galactosidase,
beta-latamase, luciferase, and alkaline phosphatase), other labels
such as chemiluminescent labels, biotinyl groups, or predetermined
polypeptide epitopes recognized by a secondary reporter (e.g.,
leucine zipper pair sequences, binding sites for secondary
antibodies, metal binding domains, and epitope tags). In some
embodiments, labels are attached by spacer arms of various lengths
to reduce potential steric hindrance.
[0046] "Fluorescent label" refers to a fluorescent moiety
incorporated onto or within a chemical structure having desirable
properties, such as binding with a target or attaching to a
polypeptide of biotinyl moieties that can be detected by avidin
(e.g., streptavidin containing a fluorescent label or enzymatic
activity that can be detected by fluorescence detection methods).
Various methods of fluorescently labeling polypeptides,
glycoproteins and other moieties are known in the art and may be
used. Examples of labels for polypeptides include, but are not
limited to dyes (e.g., FITC and rhodamine), intrinsically
fluorescent proteins, and lanthanide phosphors. In some
embodiments, labels are attached by spacer arms of various lengths
to reduce potential steric hindrance.
[0047] "Photon reducing agent" refers to a molecule or particle,
such as a colloidal particle, that reduces that amount of light
emitted from another molecule in a sample or reduces the amount of
light that excites another molecule in a sample. Typically, a
photon reducing agent reduces the amount of light emitted from
another molecule in a sample by having an absorption spectrum that
overlaps with the absorption, excitation, or emission spectrum of a
molecule that produces photons. Alternatively, some photon reducing
agents may engage in energy transfer (e.g., fluorescence resonance
energy transfer (FRET)) with a photon producing agent that prevents
or alters the emission of light from the photon producing
agent.
[0048] "Photon producing agent" refers to a molecule that can emit
photons. Typically, a photon producing agent produces photons by
absorbing light at one wavelength and emitting light of another
wavelength.
[0049] "Reporter gene" refers to a nucleotide sequence encoding a
protein that is readily detectable either by its presence or
activity, including, but not limited to, luciferase, green
fluorescent protein, chloramphenicol acetyl transferase,
beta-galactosidase, secreted placental alkaline phosphatase,
beta-lactamase, human growth hormone, and other secreted enzyme
reporters. Generally, reporter genes encode a polypeptide not
otherwise produced by a host cell, which is detectable by analysis
of the cell or a population of cells, e.g., by the direct
fluorometric, radioisotopic, optical or spectrophotometric analysis
of the cell or a population of cells and preferably without the
need to kill the cells for signal analysis. Preferably, the
reporter gene encodes an enzyme that produces a change in at least
one fluorescent property of or in the host cell. The at least one
fluorescent property is preferably detectable by qualitative,
quantitative or semi-quantitative function methods, such as the
detection of transcriptional activation. Exemplary enzymes include
esterases, phosphatases, proteases (for example, tissue plasminogen
activator or urokinase) and other enzymes (such as beta-lactamase
or luciferase or sugar hydrolases, such as beta-galactosidase)
whose function can be detected by appropriate chromogenic or
fluorogenic substrates known to those skilled in the art.
[0050] "Plate" refers to a multi-well plate, unless otherwise
modified in the context of its usage.
[0051] "Sample" refers to any fluid, solid, jelly, emulsion,
slurry, or a mixture thereof that contains a membrane compartment.
A sample is preferably an aqueous solution that contains a cell,
such as a eukaryotic cells, such as a mammalian cell, such as a
human cell.
[0052] "Signal transduction detection system" refers to a system
for detecting signal transduction across a cell membrane, typically
a cell plasma membrane. Such systems typically detect at least one
activity or physical property directly or indirectly associated
with signal transduction. For example, an activity or physical
property directly associated with signal transduction is the
activity or physical property of either the receptor (e.g., GPCR),
or a coupling protein (e.g., a Ga protein). Signal transduction
detection systems for monitoring an activity or physical property
directly associated with signal transduction, include the detection
of GTPase activity and conformational changes of members of the
signal transduction system. An activity or physical property
indirectly associated with signal transduction is the activity or
physical property produced by a molecule other than by either the
receptor (e.g., GPCR), or a coupling protein (e.g., a Ga protein)
associated with receptor (e.g., GPCR), or a coupling protein (e.g.,
a Ga protein). Such indirect activities and properties include
changes in intracellular levels of molecules (e.g., ions (e.g.,
Ca.sup.++, Na.sup.+ or K.sup.+), second messenger levels (e.g.,
cAMP, cGMP and inostol phosphate)), kinase activities,
transcriptional activity, enzymatic activity, phospholipase
activities, ion channel activities and phosphatase activities.
Signal transduction detection systems for monitoring an activity or
physical property indirectly associated with signal transduction
include, for example, transcriptional-based assays, enzymatic
assays, intracellular ion assays and second messenger assays.
[0053] "Solution fluorescence" refers to fluorescence from a
fluorophore in a solution. For instance the fluorophore may be a
test chemical (or a component associated with the test compound or
a component of the measurement system itself) in an assay buffer.
Solution fluorescence is one component of background fluorescence.
Background fluorescence may arise from other sources, such as assay
vessels (e.g., microtiter plates), optical relay systems and
backscatter.
[0054] A "target" refers to any biological entity, such as a
protein, sugar, carbohydrate, nucleic acid, lipid, a cell or
population of cells or an extract thereof, a vesicle, or any
combination thereof.
[0055] "Transmittance" refers to the fraction of incident light
that passes through a medium at a given wavelength. It can also be
considered the ratio of radiant power transmitted through a medium
to the radiant power incident on the medium at a particular
wavelength.
[0056] Other chemistry terms herein are used according to
conventional usage in the art, as exemplified by The McGraw-Hill
Dictionary of Chemical Terms (ed. Parker, S., 1985), McGraw-Hill,
San Francisco, incorporated herein by reference).
Introduction
[0057] The present invention recognizes for the first time that the
addition of a photon reducing agent can decrease undesired light
emission from a sample. Typically the sample comprises a membrane
compartment, using a photon producing agent. The present invention
also recognizes for the first time that solution fluorescence in
cell-based assays can be reduced by adding a photon reducing agent,
such as a dye, to the solution bathing the cells. Aspects of the
invention are based, in part, on the counter-intuitive finding that
the addition of a chemical having "color" can improve fluorescence
assay measurements by reducing solution fluorescence while
retaining signal fluorescence from a separate aqueous compartment.
The advantages of the present invention include: 1) increasing the
signal to noise ratio in assays utilizing membrane compartments, 2)
decreasing assay variability, 3) reducing assay time, 4) reducing
assay manipulation (especially compared to assays with washing
steps), and 5) minimizing solution fluorescence.
[0058] As a non-limiting introduction to the breadth of the
invention, the invention includes several general and useful
aspects, including: [0059] (1) a method for reducing undesirable
light emission from a sample (e.g., a biochemical or cellular
sample) with at least one photon producing agent by using at least
one photon reducing agent (e.g. dye-based photon reducing agents),
[0060] (2) a method for reducing undesirable light emission from a
sample (e.g., a biochemical or cellular sample) with at least one
photon producing agent by using at least one collisional quencher,
[0061] (3) a method for reducing undesirable light emission from a
sample (e.g., a biochemical or cellular sample) with at least one
photon producing agent by using at least one quencher, such as an
electronic quencher, [0062] (4) a method of screening test
chemicals in fluorescent assays using photon reducing agents,
[0063] (5) compositions, therapeutic compositions and kits for
practicing (1), (2), (3), (4), and (5), [0064] (6) a system for
identifying the compositions of (6), and [0065] (7) a method of
identifying a chemical with toxicological activity.
[0066] These aspects of the invention and others described herein,
can be achieved by using the methods and compositions of matter
described herein. To gain a full appreciation of the scope of the
invention, it will be further recognized that various aspects of
the invention can be combined to make desirable embodiments of the
invention. For example, the invention includes a method for
reducing background fluorescence using dye-based photon reducing
agents in assays to identify test chemicals that modulate target
proteins. Such combinations result in particularly useful and
robust embodiments of the invention.
Methods for Reducing Undesired Light Emission from a Sample Using
at Least One Photon Reducing Agent
[0067] The invention provides for a method of reducing undesirable
light emission from a sample. The method can be used with
fluorescence assays that are often hampered by solution
fluorescence that interferes with detecting a desired signal from
the sample. Cell-based assays typically emit solution fluorescence
from probes or test chemicals that can obscure desired signal
detection. To avoid these problems, the inventors devised a method
to reduce undesired light emitted from the sample by adding a
photon reducing agent to the sample. As described more fully
herein, "photon reducing agent" refers to a molecule that reduces
that amount of light from a sample by another molecule.
[0068] The method comprises the steps of contacting a sample with
at least one photon reducing agent and detecting an optical signal
from a photon producing agent. The sample typically comprises a
membrane-enclosed compartment in contact with a solid surface that
can pass (e.g. transmit) light. The membrane compartment usually
includes at least one photon producing agent. As described more
fully herein, "photon producing agent" refers to a molecule that
emits light. The photon producing agent is typically located either
within an aqueous interior of the membrane-enclosed compartment or
in association with the membrane or some other component of the
membrane compartment (e.g., a cellular organelle). The photon
reducing agent is typically located in an aqueous solution that
contacts the outer surface of the membrane compartment. The aqueous
solution that contacts the outer surface also typically contains
the source or sources of light (e.g., photon producing agents) that
lead to unwanted light emission from a sample. Photon reducing
agents reduce the light emitted from a sample that originates from
photon producing agents in the aqueous solution.
[0069] Samples in need of a reduction in undesired light emission,
as described more fully herein, are typically associated with
fluorescent assays and range from chemical or biochemical samples
(e.g., vesicles-compartmentalizing photon producing agents) to
living cell samples (e.g., cell-based assays using reporter genes).
Such samples often have solution fluorescence that contributes to
increased background fluorescence that can either prevent the
measurement of a desired signal or reduce the signal to noise ratio
compared to detection of the signal in the presence of a photon
reducing agent.
[0070] Photon producing agents can be fluorescent protein, such as
an Aequorea-related fluorescent protein or a mutant thereof (see,
for example, U.S. Pat. No. 5,625,048 to Tsien, issued Apr. 29,
1997; WO 96/23810 to Tsien et al., published Aug. 8, 1996; WO
97/28261 to Tsien et al., published Aug. 7, 1997; PCT/US97/12410 to
Tsien et al, filed Jul. 16, 1997; and PCT/US97/14593, filed Aug.
15, 1997), a fluorescent or fluorogenic enzymatic substrate (see,
for example, WO 96/30540 to Tsien et al., published Oct. 3, 1996),
a member of a FRET pair, or can detect a voltage across a membrane
of a cell (see, U.S. Pat. No. 5,661,035 to Tsien et al., issued
Aug. 26, 1997), an intracellular ion indicator, such as for calcium
ions, such as Fluo3, Fura2, Indol, or fluorescent labels used in
specific binding reactions, such as immunoassays or receptor-ligand
assays.
[0071] To reduce solution fluorescence, the invention utilizes a
photon-reducing agent. Photon reducing agents may be selected to
reduce light from another molecule by a mechanism or mechanisms
that allow for the reduction of the emission of unwanted light
emission from a sample. One class of photon reducing agents may
absorb, and therefore reduce, the amount of unwanted light emitted
from a sample comprising a photon producing agent.
[0072] Desirable photon reducing agents typically have an
absorption spectrum that overlaps with the absorption, excitation
or emission spectrum (or a combination thereof) of a photon
producing agent. Alternatively, another class of photon reducing
agents may quench (e.g., by fluorescence resonance energy transfer
(FRET)) a photon producing agent. Other quenching mechanisms or
agents may be used, including collisional quenchers, electronic
quenching, particular quenching, exeplex formation, photo-induced
electron transfer, paramagnetic or heavy-atom quenching leading to
enhanced intersystem crossing. (see generally, Principles of
Fluorescence Spectroscopy by Joseph R.
[0073] Lakowicz. Plenum Press 1983). Other photon reducing agents
are optical interferants that can reduce the amount of light
emitted from a photon producing agent by light scatter, refraction
or reflectance. For example, particulates reduce light emission
from a photon producing agent, in part, by light scattering. It is
understood that reduced light emission from a photon producing
agent can result from many types of photon reducing agents working
with different mechanisms. It is also understood that in certain
applications it will be desirable to select photon reducing agents
that reduce or decrease undesired light emission from a sample by
more than one mechanism. For instance, a photon reducing agent can
be selected that reduces solution fluorescence by FRET and has an
absorption spectrum that overlaps with the absorption, excitation
or emission spectrum of a molecule that produces light. Selection
of a photon reducing agent(s) is described more fully herein.
[0074] Typically, in a fluorescent assay, at least one photon
reducing agent can be selected that has an absorption spectrum that
overlaps with the absorption, emission or excitation spectrum of a
photon producing agent located outside of a membrane compartment.
In some instances the photon producing agent may be located inside
and outside the membrane compartment, such as with a membrane
permeable sensor that leaks out through the membrane compartment
and into the surrounding solution. A photon producing agent can
also be free or bound inside a cell, such as a living cell that
does not have a cell wall, such as a mammalian cell (such as a
human cell. An assay may also include a second photon producing
agent in an aqueous solution surrounding the membrane compartment
or at a site other than the site of desirable signal emission. The
number of photon reducing agents in an assay typically ranges from
between 1 and 5, between 1 and 4, between 1 and 3, between 1 and 2,
and may include at least two or more or at least three or more. For
example, the first photon producing agent may be a reporter gene
substrate or product located inside of a cell, and the second
photon producing agent may be a test chemical in the bathing
solution.
[0075] Photon reducing agents can be readily selected for an
absorption spectrum that overlaps with the absorption, emission or
excitation spectrum of a photon producing agent. As described
herein, the absorption spectra of a photon reducing agent can be
readily measured and compared to measured absorption, emission or
excitation spectrum of a known or expected photon producing agent.
Such known or expected photon producing agents include, fluorescent
reporter substrates, fluorescent labels, fluorescent membrane
sensors, fluorescent proteins, test chemicals and intracellular
analyte indictors (e.g., ion chelators). Methods known or developed
in the art for measuring and, comparing absorption spectra can also
be used to identify photon reducing agents. Light reducing dyes
refer to photon reducing agents that have an absorption spectrum
that overlaps with the absorption, emission or excitation spectrum
of a photon producing agent.
[0076] When selecting a photon reducing agent, such as a light
reducing dye, it is advantageous to compare the extent of its
absorption spectrum overlap with 1) the absorption, emission or
excitation spectrum of a photon producing agent in aqueous solution
and 2) the absorption, emission or excitation spectrum of the
expected signal molecule in an assay sample. This comparison can
aid in the selection of a photon reducing agent, such as a light
reducing dye, by optimizing the spectral overlaps. In addition, it
is desirable to select photon reducing agents with high extinction
coefficients in order to reduce the amount of photon reducing agent
needed for the desired effect.
[0077] Preferable photon reducing agents typically at least
partially block either or both of the excitation or emission
wavelengths of photon producing agents. In doing so, preferable
photon reducing agents reduce undesired light emission from a
sample. Such preferable photon reducing agents can be determined by
comparing the extinction coefficients of candidate photon reducing
agents with the expected photon producing agents at the desired
wavelength or range of wavelengths, by empirical observations, or
by routine experimentation to select such desired photon producing
agents using the methods of the present invention. Photon reducing
agents can reduce the emission of undesired light from a sample by
at least about 10 percent, preferably at least about 30 percent,
more preferably at least about 50, and most preferably between
about 70 and 99 percent as compared to light emission from a sample
or a particular photon producing agent in the absence of a photon
reducing agent.
[0078] Such photon producing agents and photon reducing agents can
be determined, for example, by exciting a sample comprising a
photon producing agent and a photon reducing agent with light of a
first wavelength bandwidth and collecting the emission from the
sample at a second wavelength bandwidth. Preferably, the first
wavelength bandwidth and the second wavelength bandwidth do not
overlap, but they may. Preferable first wavelength bandwidths and
preferable second wavelength bandwidths can be determined by
routine experimentation using methods of the present invention to
determine such wavelength bandwidth ranges and overlaps. Such
bandwidths preferably include the appropriate excitation or
emission peaks of at least one of the photon producing agent or
photon reducing agent, but that need not be the case because
significant excitation or emission can be obtained over a large
portion of the appropriate excitation spectra or emission
spectra.
[0079] Photon reducing agents are preferably provided at a working
concentration in a sample between about 0.1 mM and about 10 mM and
more preferably between about 0.5 mM and 5 mM. When two or more
photon reducing agents are present in a sample, the combined
concentration of the photon reducing agents is preferably between
about 0.1 mM and 10 mM and more preferably between about 0.5 mM and
5 mM. Photon reducing agents can increase the signal-to-noise
ration of an assay by between about 50% to about 100,000% or
greater, and preferably between about 500% and about 3,000%. The
percent increase in signal-to-noise ratio (S/N) in the presence of
a photon reducing agent (PRA) can be calculated by the formula
((S/N in the presence of a PRA)/(S/N in the absence of a
PRA)).times.100=percent increase in S/N.
[0080] Photon reducing agents also can be substantially impermeant
to the membrane of a membrane compartment. Substantially
impermeant, in this instance, means that under assay conditions,
the concentration of the photon reducing agent within the membrane
compartment is less than 50%, preferably less than 30%, and most
preferably less than 10% of the concentration outside the membrane
compartment.
[0081] Preferable photon reducing agents have a partition
coefficient (octanol/water) equal to or less than CCF2/AM, at a pH
between about 6 and 8, preferably about pH 7, so that the photon
reducing agent preferably partitions in an aqueous solution rather
than in a hydrophobic phase, such as a membrane (for CCF2/AM, see
U.S. Pat. No. 5,741,657 to Tsien et al., issued Apr. 21, 1998).
Also, preferable photon reducing agents have solubility in water of
at least about 1 mM and preferably at least about 10 mM under assay
conditions, such as between about 4.degree. C. and 42.degree. C.,
preferably between about 24.degree. C. and 37.degree. C. In
addition, photon reducing agents are preferably should be more
impermeant across a membrane compartment, such as a mammalian cell,
than a photon producing agent used in an assay. Photon reducing
agents can be a pH indicator dye and be dyes, such as azo dyes.
[0082] Preferable photon reducing agents also have an extinction
coefficient of between about 2,000 M.sup.-1 cm.sup.-1 to about
500,000 M.sup.-1 cm.sup.-1, preferably between about 10,000
M.sup.-1 cm.sup.-1 and 200,000 M.sup.-1 cm.sup.-1 and more
preferably greater than 10,000 M.sup.-1 cm.sup.-1 at a wavelength
or range of wavelengths used in an assay.
[0083] In many instances the photon producing agent that leads to
unwanted light emitted from the sample will be the signal molecule
in a non-desired location or compartment. Such instances typically
occur when the signal molecule is present in the surrounding
solution and reducing solution fluorescence becomes desirable. For
example, a fluorescent reporter that leaks out of a membrane
compartment, such as a cell, will often decrease the signal to
noise ratio of the assay. When the photon producing agent is the
signal molecule, it is desirable to select a light reducing dye
having an absorption spectrum that significantly overlaps with the
absorption, emission or excitation spectrum of the expected signal
molecule. Preferably, such photon reducing agents can be identified
by determining the percentage overlap of spectrum as determined
from the concentration and extinction coefficient of a photon
producing agent and a photon reducing agent at a desired wavelength
or range of wavelengths (see, Lakowicz, J. R. Principles of
Fluorescence Spectroscopy, New York: Plenum Press (1983)).
Generally, the wavelength range for such spectra is about 260 nm to
about 900 nm, although narrow ranges can be used, such as about 290
to 800 nm, and about 300 to 700 nm. In addition it is advantageous
to select a photon reducing agent with an optical density or
extinction coefficient sufficiently high to be effective in
reducing solution fluorescence.
[0084] In one embodiment of the invention, a photon producing agent
is excited with light of a narrow wavelength bandwidth, such as
light filtered through a band pass excitation filter or by
excitation with a narrow wavelength band or single wavelength of
laser light as is known in the art. The emission from the sample is
detected using a selected bandwidth of light emitted from the
photon producing agent using, for example, an emission band pass
filter. Preferably, the photon reducing agent has a high extinction
coefficient at the excitation and emission wavelengths.
[0085] Depending on the sensitivity or reactivity of the reagents
used in the assay, such as membrane compartments (such as cells) or
targets, it will be desirable to select photon reducing agents with
little or no relevant biological activity so that the photon
reducing agent does not interfere with an assay. Often it will be
valuable to test the toxicity and non-specific binding of the
photon reducing agents in the assay to insure their compatibility
with assay components. Preferably, photon reducing agents are not
toxic to cells used in a cell based assay within the time frame of
the assay. Preferably, photon reducing agents do not react with, or
bind to, the targets or other biomolecules in the assay to
undesirably alter the biological activity or property being
measured. Preferably, photon reducing agents should not cross the
(cell) membrane.
[0086] Samples can comprise one or more or two or more photon
producing agents and one or more or two or more photon reducing
agents. In the case of multiple photon producing agents or photon
reducing agents, the characteristics of the photon producing agents
or photon reducing agents can be selected to have desired
characteristics. For example, photon reducing agents can be
selected to form combinations that have absorption spectra that are
broader than the absorption spectra of the individual photon
reducing agents. These combinations of photon reducing agents can
be used to reduce the emission of undesired light from one or more
photon producing agents in a sample. Such reduced emission of
undesired light from the sample can be accomplished during the
excitation of and emission from the photon producing agent.
[0087] Light emitted from a sample can be detected by any
appropriate means for a particular assay format. For example,
fluorescence can be detected using a fluorometer, which can detect
epifluorescence. Samples can be provided in any appropriate
container from which a signal can be detected, such as vials or
wells of a microtiter plate. For microtiter plates, the number of
wells in a standard 96-well format footprint can be between about 6
and about 3,456 wells, preferably between about 96 wells and 864
wells, and more preferably between about 288 and about 384 wells,
more preferably greater than about 384 wells (see, U.S. patent
application Ser. No. 08/868,018 to Coassin et al., filed Jun. 3,
1997). Preferably, the microtiter plate has wells that have a
bottom that has at least a portion that can pass light of a
wavelength used in an assay. Membrane compartments in the sample
preferably are in contact (physical contact or optical contact)
with the bottom of such wells. In this instance, optical contact
means that the presence of a photon reducing agent, at least a
portion of the light emitted from said membrane compartment can
pass through the bottom of said well. The solution volume
containing the sample used is dependent upon the volume capacity of
the container used in an assay. Preferably, the sample volume is
between about 100 nanoliters and about 1 milliliter, preferably
between about 0.5 microliters and about 0.5 milliliter, and most
preferably between about 1 microliters and about 250 microliters or
between about 3 microliters and about 100 microliters.
[0088] The number of membrane compartments, such as cells, in a
sample is preferably between about 10 and about 1,000,000,000
membrane compartments and more preferably between about 100 and
about 200,000 membrane compartments. When the membrane compartments
are cells, the cells are preferably living, and are preferably
mammalian cells. Samples can contain a predetermined number of
cells or an unknown number of cells. Samples can contain cells that
are members of a clonal population or a heterogeneous population.
Preferably, the membrane compartments form a single layer of
membrane compartments in optical contact with an appropriate solid
surface such that the emission from a photon producing agent can
pass through the appropriate solid surface. However, the membrane
compartments may form a plurality of such layers. Membrane
compartments in optical contact with a solid surface can be in
direct contact with the solid surface, preferably between about 5
.ANG. to about the thickness of a eukaryotic cell in culture, more
preferably between about 5 .ANG. to about one-half the thickness of
a eukaryotic cell in culture, more preferably between about 10
.ANG. to about the thickness of two IgG antibodies placed Fab
region to Fab region, more preferably between about 15 .ANG. and
about the thickness of a lipid bilayer or about the thickness of a
cytoplasmic membrane of a eurkaryotic cell in culture.
[0089] Cells
[0090] Many cells can be used in the invention for cell based
assays. Such cells include, but are not limited to; baby hamster
kidney (BHK) cells (ATCC No. CCL10), mouse L cells (ATCC No.
CCLI.3), Jurkats (ATCC No. TIB 152) and 153 DG44 cells (see, Chasin
(1986) Cell. Molec. Genet. 12: 555) human embryonic kidney (HEK)
cells (ATCC No. CRL1573), Chinese hamster ovary (CHO) cells (ATCC
Nos. CRL9618, CCL61, CRL9096), PC12 cells (ATCC No. CRL17.21),
COS-7 cells (ATCC No. CRL1651) and yeast. Preferred cells for
heterologous cell surface protein expression are those that can be
readily and efficiently transfected. Preferred cells include Jurkat
cells, CHO cells, and HEK 293 cells, such as those described in
U.S. Pat. No. 5,024,939 and by Stillman et al. (1985) Mol. Cell.
Biol. 5: 2051-2060, each of which are incorporated herein by
reference.
[0091] Targets
[0092] One method of the present invention uses targets for
identifying chemicals that are useful for modulating the activity
of the target or a target having similar structural or functional
characteristics. The target can be any biological entity, such as a
protein, sugar, nucleic acid or lipid. Typically, targets will be
proteins such as enzymes or cell surface proteins. Targets can be
assayed in either biochemical assays (targets free of cells) or
cell based assays (targets associated with a cell).
[0093] For example, cells may be loaded with ion or voltage
sensitive dyes to report receptor or ion channel activity, such as
calcium channels, or N-methyl-D-aspartate (NMDA) receptors, GABA
receptors, kainate/AMPA receptors, nicotinic acetylcholine
receptors, sodium channels, calcium channels, potassium channels,
excitatory amino acid (EAA) receptors, and nicotinic acetylcholine
receptors. Assays for determining activity of such receptors can
also use agonists and antagonists to use as negative or positive
controls to assess the activity of tested chemicals. In preferred
embodiments of automated assays for identifying chemicals that have
the capacity to modulate the function of receptors or ion channels
(e.g., agonists, antagonists), changes in the level of ions in the
cytoplasm or membrane voltage will be monitored using an
ion-sensitive or membrane voltage fluorescent indicator,
respectively. Among the ion-sensitive indicators and voltage probes
that may be employed are those disclosed in the Molecular Probes
1997 Catalog, herein incorporated by reference.
[0094] Other methods of the present invention concern determining
the activity of receptors. Receptor activation can sometimes
initiate subsequent intracellular events that release intracellular
stores of calcium ions for use as a second messenger or the influx
of calcium ions into a cell. Activation of some G-protein-coupled
receptors stimulates the formation of inositol triphosphate (IP3 a
G-protein coupled receptor or tyrosine kinase second messenger)
through phospholipase C-- mediated hydrolysis of
phosphatidylinositol, Berridge and Irvine (1984), Nature 312:
315-21. IP3 in turn stimulates the release of intracellular calcium
ion stores. Thus, a change in cytoplasmic calcium ion levels caused
by the release of calcium ions from intracellular stores can be
used to reliably determine G-protein-coupled receptor function.
Among G-protein-coupled receptors are muscarinic acetylcholine
receptors (mAChR), adrenergic receptors, serotonin receptors,
dopamine receptors, angiotensin receptors, adenosine receptors,
bradykinin receptors, metabotropic excitatory amino acid receptors,
and the like. Cells expressing such G-protein-coupled receptors may
exhibit increased cytoplasmic calcium levels as a result of
contribution from both intracellular stores and via activation of
ion channels. In such instances, it may be desirable, although not
necessary, to conduct such assays in calcium-free buffer,
optionally supplemented with a chelating agent such as EGTA, to
distinguish fluorescence response resulting from calcium release
from internal stores.
[0095] Exemplary membrane proteins that may be targets include, but
are not limited to, surface receptors and ion channels. Surface
receptors include, but are not limited to, muscarinic receptors,
e.g., human M2 (GenBank accession #M16404); rat M3 (GenBank
accession #M16407), human M4 (GenBank accession #M16405), human M5
(Bonner, et al., (1988) Neuron 1, pp. 403-410); and the like.
Neuronal nicotinic acetylcholine receptors include, e.g., the human
.alpha.2, .alpha.3, and .beta.2, subtypes, the human .alpha.5
subtype (Chini, et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89:
1572-1576), the rat .alpha.2 subunit (Wada, et al. (1988) Science
240, pp. 330-334), the rat .alpha.3 subunit (Boulter, et al. (1986)
Nature 319, pp. 368-374), the rat .alpha.4 subunit (Goldman, et al.
(1987) Cell 48, pp. 965-973), the rat .alpha.5 subunit (Boulter, et
al. (1990) I. Biol. Chem. 265, pp. 4472-4482), the chicken .alpha.7
subunit (Couturier et al. (1990) Neuron 5: 847-856), the rat
.beta.2 subunit (Deneris, et al. (1988) Neuron 1, pp. 45-54), the
rat .beta.3 subunit (Deneris, et al. (1989) J. Biol. Chem. 264, pp.
6268-6272), the rat .beta.4 subunit (Duvoisin, et al. (1989) Neuron
3, pp. 487-496), combinations of the rat .alpha. subunits, .beta.
subunits and a and p subunits. GABA receptors include, e.g., the
bovine n, and p, subunits (Schofield, et al. (1987) Nature 328, pp.
221-227), the bovine n, and a, subunits (Levitan, et al. (1988)
Nature 335, pp. 76-79), the .gamma.-subunit (Pritchett, et al.
(1989) Nature 338, pp. 582-585), the p, and p, subunits (Ymer, et
al. (1989) EMBO J. 8, pp. 1665-1670), the 6 subunit (Shivers, B. D.
(1989) Neuron 3, pp. 327-337), and the like. Glutamate receptors
include, e.g., rat GluR1 receptor (Hollman, et al. (1989) Nature
342, pp. 643-648), rat GluR2 and GluR3 receptors (Boulter et al.
(1990) Science 249:1033-1037, rat GluR4 receptor (Keinanen et al.
(1990) Science 249: 556-560), rat GluR5 receptor (Bettler et al.
(1990) Neuron 5: 583-595), rat GluR6 receptor (Egebjerg et al.
(1991) Nature 351: 745-748), rat GluR7 receptor (Bettler et al.
(1992) neuron 8:257-265), rat NMDAR1 receptor (Moriyoshi et al.
(1991) Nature 354:31-37 and Sugihara et al. (1992) Biochem.
Biophys. Res. Comm. 185:826-832), mouse NMDA el receptor (Meguro et
al. (1992) Nature 357: 70-74), rat NMDAR2A, NMDAR2B and NMDAR2c
receptors (Monyer et al. (1992) Science 256: 1217-1221), rat
metabotropic mGluR1 receptor (Houamed et al. (1991) Science 252:
1318-1321), rat metabotropic mGluR2, mGluR3 and mGluR4 receptors
(Tanabe et al. (1992) Neuron 8:169-179), rat metabotropic mGluR5
receptor (Abe et al. (1992) I. Biol. Chem. 267: 13361-13368), and
the like. Adrenergic receptors include, e.g., human pl (Frielle, et
al. (1987) Proc. Natl. Acad. Sci. 84, pp. 7920-7924), human
.alpha.2 (Kobilka, et al. (1987) Science 238, pp. 650-656), hamster
.beta.2 (Dixon, et al. (1986) Nature 321, pp. 75-79), and the like.
Dopamine receptors include, e.g., human D2 (Stormann, et al. (1990)
Molec. Pharm. 37, pp. 1-6), mammalian dopamine D2 receptor (U.S.
Pat. No. 5,128,254), rat (Bunzow, et al. (1988) Nature 336, pp.
783-787), and the like. NGF receptors include, e.g., human NGF
receptors (Johnson, et al. (1986) Cell 47, pp. 545-554), and, the
like. Serotonin receptors include, e.g., human 5HT1a (Kobilka, et
al. (1987) Nature 329, pp. 75-79), serotonin 5HT1C receptor (U.S.
Pat. No. 4,985,352), human 5HT1D (U.S. Pat. No. 5,155,218), rat
5HT2 (Julius, et al. (1990) PNAS 87, pp. 928-932), rat 5HT1c
(Julius, et al. (1988) Science 241, pp. 558-564), and the like.
[0096] Ion channels include, but are not limited to, calcium
channels comprised of the human calcium channel .alpha.2 .beta.
and/or .gamma.-subunits (see WO89/09834; human neuronal .alpha.2
subunit), rabbit skeletal muscle al subunit (Tanabe, et al. (1987)
Nature 328, pp. 313-E318), rabbit skeletal muscle .alpha.2 subunit
(Ellis, et al. (1988) Science 241, pp. 1661-1664), rabbit skeletal
muscle p subunit (Ruth, et al. (1989) Science 245, pp. 1115-1118),
rabbit skeletal muscle .gamma. subunit (Jay, et al. (1990) Science
248, pp. 490-492), and the like. Potassium ion channels include,
e.g., rat brain (BK2) (McKinnon, D. (1989) J. Biol Chem. 264, pp.
9230-8236), mouse brain (BK1) (Tempel, et al. (1988) Nature 332,
pp. 837-839), and the like. Sodium ion channels include, e.g., rat
brain I and E (Noda, et al. (1986) Nature 320, pp. 188-192), rat
brain III (Kayano, et al. (1988) FEBS Lett. 228, pp. 187-1.94),
human II (ATCC No. 59742, 59743 and Genomics 5: 204-208 (1989),
chloride ion channels (Thiemann, et al. (1992), Nature 356, pp.
57-60 and Paulmichl, et al. (1992) Nature 356, pp. 238-241), and
others known or developed in the art.
[0097] Intracellular receptors may also be used as targets, such as
estrogen receptors, glucocorticoid receptors, androgen receptors,
progesterone receptors, and mineralocorticoid receptors, in the
invention. Transcription factors and kinases can also be used as
targets, as well as plant targets.
[0098] Various methods of identifying activity of a chemical with
respect to a target can be applied, including: ion channels (PCT
publication WO 93/13423), intracellular receptors (PCT publication
WO 96/41013), U.S. Pat. No. 5,548,063, U.S. Pat. No. 5,171,671,
U.S. Pat. No. 5,274,077, U.S. Pat. No. 4,981,784, EP 0 540 065 A1,
U.S. Pat. No. 5,071,773, and U.S. Pat. No. 5,298,429. Fluorescent
assays that can be used with the invention include those described
in PCT WO 96/3540 (Tsien), PCT WO 96/41166 (Tsien) and PCT WO
96/23810 (Tsien). The methods set forth in PCT WO 96/3540 (Tsien)
and PCT WO 96/23810 (Tsien) can also be combined with methods
described in U.S. Pat. Nos. 5,401,629 and 5,436,128 by Harpold et
al. for assays of cell surface receptors and the cell based
intracellular receptor assays referenced herein. All of the
foregoing references are herein incorporated by reference in their
entirety.
[0099] Fluorescence Measurements
[0100] When using fluorescent sensors, indicators or probes such as
photon producing agents, it will be recognized that different types
of fluorescent monitoring systems can be used to practice the
invention. Preferably, FACS systems or systems dedicated to high
throughput screening, e.g 96 well or greater microtiter plates or
multi-well platforms are used to identify compounds such as
therapeutic compounds and to assess the toxicology of such
compounds (see U.S. application Ser. No. 08/858,016 to Styli et al,
filed May 16, 1997). Such high throughput screening systems can
comprise, for example: [0101] a) a storage and retrieval module for
storing a plurality of chemicals in solution in addressable
chemical wells, a chemical well retriever, and having programmable
selection and retrieval of said addressable chemical wells, and
having a storage capacity for at least 100,000 said addressable
wells, [0102] wherein at least one of said addressable wells
comprises a photon reducing agent, [0103] b) a sample distribution
module comprising a liquid handler to aspirate or dispense
solutions from selected said addressable chemical wells, said
chemical distribution module having programmable selection of, and
aspiration from, said selected addressable chemical wells and
programmable dispensation into selected addressable sample wells,
and said liquid handler can dispense into arrays of addressable
wells with different densities of addressable wells per centimeter
squared, [0104] c) a sample transporter to transport said selected
addressable chemical wells to said sample distribution module and
optionally having programmable control of transport of said
selected addressable chemical wells, [0105] d) a reaction module
comprising either a reagent dispenser to dispense reagents into
said selected addressable sample wells or a fluorescent detector to
detect chemical reactions ins said selected addressable sample
wells, and [0106] e) a data processing and integration module,
[0107] wherein said storage and retrieval module, said sample
distribution module, and said reaction module are integrated and
programmably controlled by said data processing and integration
module; and said storage and retrieval module, said sample
distribution module, said sample transporter, said reaction module
and said data processing and integration module are operably linked
to facilitate rapid processing of said addressable sample
wells.
[0108] Multi-well platforms useful in the present invention can
have between about 6 and about 5,000 wells, preferably between
about 96 and about 4,000 wells, most preferably in multiples of 96
(see U.S. patent application Ser. No. 08/867,567, filed Jun. 2,
1997; U.S. patent application Ser. No. 08/868,018, filed Jun. 3,
1997; U.S. patent application Ser. No. 08/867,584, filed Jun. 2,
1997; U.S. patent application Ser. No. 08/868,049, filed Jun. 3,
1997; U.S. patent application Ser. No. 09/030,578, filed Feb. 24,
1998; and U.S. patent application Ser. No. 09/028,283, filed Feb.
24, 1998). Methods of performing assays on fluorescent materials
are well known in the art and are described in, e.g., Lakowicz, J.
R., Principles of Fluorescence Spectroscopy, New York: Plenum Press
(1983); Herman, B., Resonance energy transfer microscopy; in:
Fluorescence Microscopy of Living Cells in Culture, Part B, Methods
in Cell Biology, vol. 30, ed. Taylor, D. L. & Wang, Y.-L., San
Diego: Academic Press (1989), pp. 219-243: Turro, N.J., Modern
Molecular Photochemistry, Menlo Park: Benjamin-Cummings Publishing
Co. Inc. (1978), pp. 296-361.
[0109] The present invention can be used to increase the
signal-to-noise ratio in fluorescence activated cell sorting
(FACs). In this aspect of the invention, the membrane compartment
comprises at least one photon producing agent and the surrounding
solution exhibits unwanted optical background (such as
fluorescence) from, for example, at least one photon producing
agent. In this embodiment of the invention, the sample volume is
preferably a small droplet comprising the membrane compartment,
such as are useful in FACs analysis. The optical path length
through the droplet is preferably only a few micrometers, so that
the reduction of the excitation light or absorption of the emitted
fluorescence by absorptive filtering in the droplet is small.
Significant reduction of unwanted solution fluorescence from the
droplet can be achieved under such conditions when the photon
reducing agent in the droplet can interact with the excited state
of the molecule which is the source of the unwanted solution
fluorescence. Interactions that give rise to such beneficial
reduction in unwanted solution fluorescence include, but are not
limited to, fluorescence resonance energy transfer, collision
quenching, ground state dark complex formation, paramagnetic
enhancement of intersystem crossing, Dexter exchange coupling,
photo-induced electron transfer. The common property of these
interactions is that they occur over molecular distances of less
than about 20 nm, and comprise a form of energy transfer other than
simple absorption due to inner filtering. The particular conditions
for this aspect of the invention can be determined using routine
experimentation using the methods of the present invention.
[0110] Fluorescence in a sample can be measured using a
fluorimeter. In general, excitation radiation from an excitation
source having a first wavelength passes through excitation optics.
The excitation optics allow the excitation radiation to excite the
sample. In response, fluorescent probes in the sample emit
radiation that has a wavelength that is different from the
excitation wavelength. Collection optics then collect the emission
from the sample. The device can include a temperature controller to
maintain the sample at a specific temperature while it is being
measured. According to one embodiment, a multi-axis translation
stage moves a microtiter plate holding a plurality of samples in
order to position different wells to be exposed. The multi-axis
translation stage, temperature controller, auto-focusing feature,
and electronics associated with imaging and data collection can be
managed by an appropriately programmed digital computer. The
computer also can transform the data collected during the assay
into another format for presentation.
[0111] Preferably, fluorescence resonance energy transfer (FRET),
can be used as a way of monitoring activity inside a cell, such as
with the reporter gene system described in Tsien et al (PCT
WO96/30540). The degree of FRET can be determined by any
appropriate spectral or fluorescence lifetime characteristic of the
excited construct. For example, the degree of FRET can be measured
by determining the intensity of the fluorescent signal from the
donor, the intensity of fluorescent signal from the acceptor, the
ratio of the fluorescence amplitudes near the acceptor's emission
maxima to the fluorescence amplitudes near the donor's emission
maximum, or the excited state lifetime of the donor. For example,
cleavage of the linker increases the intensity of fluorescence from
the donor, decreases the intensity of fluorescence from the
acceptor, increases the ratio of fluorescence amplitudes from the
donor to that from the acceptor, and increases the excited state
lifetime of the donor.
[0112] As would be readily appreciated by those skilled in the art,
the efficiency of fluorescence resonance energy transfer depends on
the fluorescence quantum yield of the donor fluorophore, the
orientation of the fluorophore, the donor-acceptor distance, and
the overlap integral of donor fluorescence emission and acceptor
absorption. The energy transfer is most efficient when a donor
fluorophore with high fluorescence quantum yield (preferably, one
approaching 100%) is paired with an acceptor with a large
extinction coefficient at wavelengths coinciding with the emission
of the donor. The dependence of fluorescence energy transfer on the
above parameters has been reported (Forster, T. (1948) Ann. Physik
2: 55-75; Lakowicz, J. R., Principles of Fluorescence Spectroscopy,
New York: Plenum Press (1983); Herman, B., Resonance energy
transfer microscopy, in: Fluorescence Microscopy of Living Cells in
Culture, Part B, Methods in Cell Biology, Vol 30, ed. Taylor, D. L.
& Wang, Y.-L., San Diego: Academic Press (1989), pp. 219-243;
Turro, N. J., Modern Molecular Photochemistry, Menlo Part:
Benjamin/Cummings Publishing Co., Inc. (1978), pp. 296-361). Also,
tables of spectral overlap integrals are readily available to those
working in the field (for example, Beriman, I. B. Energy transfer
parameters of aromatic compounds, Academic Press, New York and
London (1973)). The distance between the donor and acceptor at
which FRET occurs with 50% efficiency is termed R.sub.0 and can be
calculated from the spectral overlap integrals. For the
donor-acceptor pair fluorescein-tetramethyl rhodamine, which is
frequently used for distance measurement in proteins, this distance
R.sub.0 is around 50-70 .ANG. (dos Remedios, C. G. et al. (1987) J.
Muscle Research and Cell Motility 8:97-117). The distance at which
the energy transfer in this pair exceeds 90% is about 45 .ANG..
[0113] Preferably, changes in the degree of FRET are determined as
a function of the change in the ratio of the amount of fluorescence
from the donor and acceptor, an analysis process referred to as
"ratioing." Changes in the absolute amount of substrate, excitation
intensity, and turbidity or other background absorbances in the
sample at the excitation wavelength affect the intensities of
fluorescence from both the donor and acceptor approximately in
parallel. Therefore, the ratio of the two emission intensities is a
preferred and more robust measure of cleavage than either intensity
alone.
[0114] The excitation state lifetime of the donor moiety is,
likewise, independent of the absolute amount of substrate,
excitation intensity, or turbidity or other background absorbances.
Its measurement requires equipment with nanosecond time resolution,
except in the special case of transition metal complexes, such as
lanthanide complexes, in which case microsecond to millisecond
resolution is sufficient.
[0115] The ratiometric fluorescent reporter system described herein
has significant advantages over existing reporters for gene
integration analysis, as it allows sensitive detection and
isolation of both expressing and non-expressing single living
cells. This assay system uses a non-toxic, non-polar fluorescent
substrate that is easily loaded and then trapped intracellularly.
Cleavage of the fluorescent substrate by beta-lactamase yields a
fluorescent emission shift as substrate is converted to product.
Because the beta-lactamase reporter readout is ratiometric, it is
unique among reporter gene assays because it controls for variables
such as the amount of substrate loaded into individual cells. The
stable, easily detected, intracellular readout eliminates the need
for establishing clonal cell lines prior to expression
analysis.
Method of Screening Test Chemicals in Fluorescent Assays Using at
Least One Photon Reducing Agent
[0116] The present invention also includes a method of identifying
a chemical with a biological activity (including toxicological
activities). The method is performed by contacting a sample with a
test chemical, wherein the sample comprises a target and a photon
producing agent. The sample is also contacted with at least one
photon reducing agent and the optical signal from the photon
producing agent is detected. The photon reducing agent may be added
before or after the test chemical, as appropriate. The sample can
include a membrane compartment in contact with a solid surface that
can pass light. The membrane compartment includes at least one
photon producing agent that directly or indirectly monitors the
activity of the target. The photon reducing agent is in an aqueous
solution that contacts the outer surface of said membrane
compartment. Preferably, the photon reducing agent has an
absorption spectra that overlaps with the absorption, emission or
excitation spectrum of the photon producing agent or of a second
photon producing agent in said aqueous solution. Also, the first
photon producing agent can, in some instances, transfer
fluorescence resonance energy to the photon reducing agent.
Alternatively, the second photon producing agent in an aqueous
solution can transfer fluorescence resonance energy to the photon
reducing agent. The present invention also includes a therapeutic
compound or composition identified by this method.
[0117] A test chemical with a biological activity, such as a
therapeutic, can be identified by contacting a test chemical
suspected of having a biological activity with a sample comprising
a membrane compartment comprising a target, such as a mammalian
cell comprising a receptor. In some assays, the binding of a test
chemical with a target can result in the expression of a reporter
gene in the membrane compartment. The reporter gene can encode a
photon producing agent or precursor photon producing agent, or
encode an enzyme that can produce a photon producing agent from an
appropriate substrate. If the sample contains a test chemical with
a biological activity reported by the reporter gene, then the
amount of a fluorescent reporter product in the sample, such as
inside or outside of the cell, will either increase or decrease
relative to background or control levels. The amount of the
fluorescent reporter product is measured by exciting the
fluorescent reporter product with an appropriate radiation of a
first wavelength and measuring the emission of radiation of a
second wavelength emitted from said sample. The amount of emission
is compared to background or control levels of emission. If the
sample having the test chemical exhibits increased or decreased
emission relative to that of the control or background levels, then
a candidate modulator has been identified. The amount of emission
is related to the amount or potency of the therapeutic in the
sample. Such methods are described in, for example, Tsien
(PCT/US90/04059). Such methods identify candidate modulators of
biological processes. The candidate modulator can be further
characterized and monitored for structure, potency, toxicology, and
pharmacology using well known methods or those described
herein.
[0118] The signal-to-noise ratio of such assays can be increased by
the addition of at least one photon reducing agent during the
course of such assays. The addition of at least one photon reducing
agent can also increase the sensitivity and precision of such
assays.
[0119] The structure of a candidate modulator identified by the
invention can be determined or confirmed by methods known in the
art, such as mass spectroscopy and nuclear magnetic resonance
(NMR). For putative chemicals with a biological activity stored for
extended periods of time, the structure, activity, and potency of
candidate modulators can be confirmed.
[0120] Depending on the system used to identify candidate
modulators, a candidate modulator can have putative pharmacological
activity. For example, if the candidate modulator is found to
inhibit T-cell proliferation (activation) in vitro, then the
candidate modulator would have presumptive pharmacological
properties as an immunosuppressant or anti-inflammatory (see,
Suthanthiran et al., Am. J. Kidney Disease, 28:159-172 (1996)).
Such nexuses are known in the art for several disease states, and
more are expected to be discovered over time. Based on such
nexuses, appropriate confirmatory in vitro and in vivo models of
pharmacological activity, as well as toxicology, can be selected.
The methods described herein can also be used to assess
pharmacological selectivity and specificity, and toxicity.
[0121] Bioavailability and Toxicology of Candidate Modulators
[0122] Once identified, candidate modulators can be evaluated for
bioavailability and toxicological effects using known methods (see,
Lu, Basic Toxicology, Fundamentals, Target Organs, and Risk
Assessment, Hemisphere Publishing Corp., Washington (1985); U.S.
Pat. No. 5,196,313 to Culbreth (issued Mar. 23, 1993) and U.S. Pat.
No. 5,567,952 to Benet (issued Oct. 22, 1996). For example,
toxicology of a candidate modulator can be established by
determining in vitro toxicity towards a cell line, such as a
mammalian i.e. human, cell line. Candidate modulators can be
treated with, for example, tissue extracts, such as preparations of
liver, such as microsomal preparations, to determine increased or
decreased toxicological properties of the chemical after being
metabolized by a whole organism. The results of these types of
studies are often predictive of toxicological properties of
chemicals in animals, such as mammals, including humans.
[0123] Such bioavailability and toxicological methods can be
performed using the methods, preferable using the screening systems
of the present invention. Such methods include contacting a sample
having a target with at least one photon producing agent, at least
one photon reducing agent, and a test chemical. An optical signal
from said at least one photon producing agent is detected, wherein
said optical signal is related to a toxicological activity.
Bioavailability is any known in the art and can be detected, for
example by measuring reporter genes that are activated during
bioavailability criteria. Toxicological activity is any known in
the art, such as apoptosis, cell lysis, crenation, cell death and
the like. The toxicological activity can be measured using reporter
genes that are activated during toxicological activity or by cell
lysis (see WO 98/13353, published Apr. 2, 1998). Preferred reporter
genes produce a fluorescent or luminescent translational product
(such as, for example, a Green Fluorescent Protein (see, for
example, U.S. Pat. No. 5,625,048 to Tsien et al., issued Apr. 29,
1998; U.S. Pat. No. 5,777,079 to Tsien et al., issued Jul. 7, 1998;
WO 96/23810 to Tsien, published 8/8/96; WO 97/28261, published
8/7/97; PCT/US97/12410, filed Jul. 16, 1997; PCT/US97/14595, filed
Aug. 15, 1997)) or a translational product that can produce a
fluorescent or luminescent product (such as, for example,
beta-lactamase (see, for example, U.S. Pat. No. 5,741,657 to Tsien,
issued Apr. 21, 1998, and WO 96/30540, published 10/3/96)), such as
an enzymatic degradation product. Cell lysis can be detected in the
present invention as a reduction in a fluorescence signal from at
least one photon producing agent within a cell in the presence of
at least one photon reducing agent. Such toxicological
determinations can be made using prokaryotic or eukaryotic cells,
optionally using toxicological profiling, such as described in
PCT/US94/00583, filed Jan. 21, 1994, German Patent No
69406772.5-08, issued Nov. 25, 1997; EPC 0680517, issued Nov. 12,
1994; U.S. Pat. No. 5,589,337, issued Dec. 31, 1996; EPO 651825,
issued Jan. 14, 1998; and U.S. Pat. No. 5,585,232, issued Dec. 17,
1996)
[0124] Alternatively, or in addition to these in vitro studies, the
bioavailability and toxicological properties of a candidate
modulator in an animal model, such as mice, rats, rabbits, or
monkeys, can be determined using established methods (see, Lu,
supra (1985): and Creasey, Drug Disposition in Humans. The Basis of
Clinical Pharmacology, Oxford University Press, Oxford (1979),
Osweiler, Toxicology, Williams and Wilkins, Baltimore, Md. (1995),
Yang, Toxicology of Chemical Mixtures; Case Studies, Mechanisms,
and Novel Approaches, Academic Press, Inc., San Diego, Calif.
(1994), Burrell et al., Toxicology of the Immune System; A Human
Approach, Van Nostrand Reinhld, Co. (1997), Niesink et al.,
Toxicology; Principles and Applications, CRC Press. Boca Raton.
Fla. (1996)). Depending on the toxicity, target organ, tissue,
locus, and presumptive mechanism of the candidate modulator, the
skilled artisan would not be burdened to determine appropriate
doses, LD.sub.50 values, routes of administration, and regimes that
would be appropriate to determine the toxicological properties of
the candidate modulator. In addition to animal models, human
clinical trials can be performed following established procedures,
such as those set forth by the United States Food and Drug
Administration (USFDA) or equivalents of other governments. These
toxicity studies provide the basis for determining the efficacy of
a candidate modulator in vivo.
Efficacy of Candidate Modulators
[0125] Efficacy of a candidate modulator can be established using
several art recognized methods, such as in vitro methods, animal
models, or human clinical trials (see, Creasey, supra (1979)).
Recognized in vitro models exist for several diseases or
conditions. For example, the ability of a chemical to extend the
life-span of HIV-infected cells in vitro is recognized as an
acceptable model to identify chemicals expected to be efficacious
to treat HIV infection or AIDS (see, Daluge et al., Antimicro.
Agents Chemother. 41:1082-1093 (1995)). Furthermore, the ability of
cyclosporin A (CsA) to prevent proliferation of T-cells in vitro
has been established as an acceptable model to identify chemicals
expected to be efficacious as immunosuppressants (see, Suthanthiran
et al., supra, (1996)). For nearly every class of therapeutic,
disease, or condition, an acceptable in vitro or animal model is
available. Such models exist, for example, for gastro-intestinal
disorders, cancers, cardiology, neurobiology, and immunology. In
addition, these in vitro methods can use tissue extracts, such as
preparations of liver, such as microsomal preparations, to provide
a reliable indication of the effects of metabolism on the candidate
modulator. Similarly, acceptable animal models may be used to
establish efficacy of chemicals to treat various diseases or
conditions. For example, the rabbit knee is an accepted model for
testing chemicals for efficacy in treating arthritis (see, Shaw and
Lacy, J. Bone Joint Surg. (Br) 55:197-205 (1973)). Hydrocortisone,
which is approved for use in humans to treat arthritis, is
efficacious in this model which confirms the validity of this model
(see, McDonough, Phys. Ther. 62:835-839 (1982)). When choosing an
appropriate model to determine efficacy of a candidate modulator,
the skilled artisan can be guided by the state of the art to choose
an appropriate model, dose, and route of administration, regime,
and endpoint and as such would not be unduly burdened In addition
to animal models, human clinical trials can be used to determine
the efficacy of a candidate modulator in humans. The USFDA, or
equivalent governmental agencies, have established procedures for
such studies (see www.fda.gov).
[0126] Selectivity of Candidate Modulators
[0127] The in vitro and in vivo methods described above also
establish the selectivity of a candidate modulator. It is
recognized that chemicals can modulate a wide variety of biological
processes or be selective. Panels of cells based on the present
invention can be used to determine the specificity of the candidate
modulator. Selectivity is evident, for example, in the field of
chemotherapy, where the selectivity of a chemical to be toxic
towards cancerous cells, but not towards non-cancerous cells, is
obviously desirable. Selective modulators are preferable because
they have fewer side effects in the clinical setting. The
selectivity of a candidate modulator can be established in vitro by
testing the toxicity and effect of a candidate modulator on a
plurality of cell lines that exhibit a variety of cellular pathways
and sensitivities. The data obtained from these in vitro toxicity
studies can be extended animal model studies, including human
clinical trials, to determine toxicity, efficacy, and selectivity
of the candidate modulator.
[0128] Identified Compositions
[0129] The invention includes compositions such as novel chemicals,
and therapeutics identified as having activity by the operation of
methods, systems or components described herein. Novel chemicals,
as used herein, do not include chemicals already publicly known in
the art as of the filing date of this application. Typically, a
chemical would be identified as having activity from using the
invention and then its structure revealed from a proprietary
database of chemical structures or determined using analytical
techniques such as mass spectroscopy.
[0130] One embodiment of the invention is a chemical with useful
activity, comprising a chemical identified by the method described
above. Such compositions include small organic molecules, nucleic
acids, peptides and other molecules readily synthesized by
techniques available in the art and developed in the future. For
example, the following combinatorial compounds are suitable for
screening: peptoids (PCT Publication No. WO 91/19735, 26 Dec.
1991), encoded peptides (PCT Publication No. WO 93/20242, 14 Oct.
1993), random bio-oligomers (PCT Publication WO 92/00091, 9 Jan.
1992), benzodiazepines (U.S. Pat. No. 5,288,514), diversomeres such
as hydantoins, benzodiazepines and dipeptides (Hobbs DeWitt, S. et
al, Proc. Nat. Acad. Sci. USA 90: 6909-6913 (1993)), vinylogous
polypeptides (Hagihara et al., J. Amer. Chem. Soc. 114: 6568
(1992)), nonpeptidal peptidomimetics with a Beta-D-Glucose
scaffolding (Hirschmann, R. et al., J. Amer. Chem. Soc. 114:
9217-9218 (1992)), analogous organic syntheses of small compound
libraries (Chen, C. et al., J. Amer. Chem. Soc. 116:2661 (1994)),
oligocarbamates (Cho, C. Y. et al., Science 261: 1303 (1993)),
and/or peptidyl phosphonates (Campbell, D. A. et al., J. Org. Chem.
59: 658 (1994)). See, generally, Gordon, E. M. et al., J. Med.
Chem. 37: 1385 (1994). The contents of all of the aforementioned
publications are incorporated herein by reference.
[0131] The present invention also encompasses the identified
chemicals and their respective compositions, typically in a
pharmaceutical composition of the present invention that comprise a
pharmaceutically acceptable carrier prepared for storage and
subsequent administration, which have the pharmaceutically
effective amount of the products disclosed above in a
pharmaceutically acceptable carrier or diluent. Acceptable carriers
or diluents for therapeutic use are well known in the
pharmaceutical art, and are described, for example, in Remington's
Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit.
1985). Preservatives, stabilizers, dyes and even flavoring agents
may be provided in the pharmaceutical composition. For example,
sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid
may be added as preservatives. In addition, antioxidants and
suspending agents may be used.
[0132] The compositions of the present invention may be formulated
and used as tablets, capsules or elixirs for oral administration;
suppositories for rectal administration; sterile solutions,
suspensions for injectable administration; and the like.
Injectables can be prepared in conventional forms, either as liquid
solutions or suspensions, solid forms suitable for solution or
suspension in liquid prior to injection, or as emulsions. Suitable
excipients are, for example, water, saline, dextrose, mannitol,
lactose, lecithin, albumin, sodium glutamate, cysteine
hydrochloride, and the like. In addition, if desired, the
injectable pharmaceutical compositions may contain minor amounts of
nontoxic auxiliary substances, such as wetting agents, pH buffering
agents, and the like. If desired, absorption enhancing preparations
(e.g., liposomes), may be utilized.
[0133] The pharmaceutically effective amount of the composition
required as a dose will depend on the route of administration, the
type of animal being treated, and the physical characteristics of
the specific animal under consideration. The dose can be tailored
to achieve a desired effect, but will depend on such factors as
weight, diet, concurrent medication and other factors which those
skilled in the medical arts will recognize.
[0134] In practicing the methods of the invention, the products or
compositions can be used alone or in combination with one another,
or in combination with other therapeutic or diagnostic agents.
These products can be utilized in vivo, ordinarily in a mammal,
preferably in a human, or in vitro. In employing them in vivo, the
products or compositions can be administered to the mammal in a
variety of ways, including parenterally, intravenously,
subcutaneously, intramuscularly, colonically, rectally, nasally or
intraperitoneally, employing a variety of dosage forms. Such
methods may also be applied to testing chemical activity in
vivo.
[0135] As will be readily apparent to one skilled in the art, the
useful in vivo dosage to be administered and the particular mode of
administration will vary depending upon the age, weight and
mammalian species treated, the particular compounds employed, and
the specific use for which these compounds are employed. The
determination of effective dosage levels, that is the dosage levels
necessary to achieve the desired result, can be accomplished by one
skilled in the art using routine pharmacological methods.
Typically, human clinical applications of products are commenced at
lower dosage-levels, with dosage level being increased until the
desired effect is achieved. Alternatively, acceptable in vitro
studies can be used to establish useful doses and routes of
administration of the compositions identified by the present
methods using established pharmacological methods.
[0136] In non-human animal studies, applications of potential
products are commenced at higher dosage levels, with dosage being
decreased until the desired effect is no longer achieved or adverse
side effects disappear. The dosage for the products of the present
invention can range broadly depending upon the desired affects and
the therapeutic indication. Typically, dosages may be between about
10 kg/kg and 100 mg/kg body weight, preferably between about 100
.mu.g/kg and 10 mg/kg body weight. Administration is preferably
oral on a daily basis.
[0137] The exact formulation, route of administration and dosage
can be chosen by the individual physician in view of the patient's
condition. (See e.g., Fingl et al., in The Pharmacological Basis of
Therapeutics, 1975). It should be noted that the attending
physician would know how to and when to terminate, interrupt, or
adjust administration due to toxicity, or to organ dysfunctions.
Conversely, the attending physician would also know to adjust
treatment to higher levels if the clinical response were not
adequate (precluding toxicity). The magnitude of an administrated
dose in the management of the disorder of interest will vary with
the severity of the condition to be treated and to the route of
administration. The severity of the condition may, for example, be
evaluated, in part, by standard prognostic evaluation methods.
Further, the dose and perhaps dose frequency, will also vary
according to the age, body weight, and response of the individual
patient. A program comparable to that discussed above may be used
in veterinary medicine.
[0138] Depending on the specific conditions being treated, such
agents may be formulated and administered systemically or locally.
Techniques for formulation and administration may be found in
Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co.,
Easton, Pa. (1990). Suitable routes may include oral, rectal,
transdermal, vaginal, transmucosal, or intestinal administration;
parenteral delivery, including intramuscular, subcutaneous,
intramedullary injections, as well as intrathecal, direct
intraventricular, intravenous, intraperitoneal, intranasal, or
intraocular injections.
[0139] For injection, the agents of the invention may be formulated
in aqueous solutions, preferably in physiologically compatible
buffers such as Hanks' solution, Ringer's solution, or
physiological saline buffer. For such transmucosal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art.
Use of pharmaceutically acceptable carriers to formulate the
compounds herein disclosed for the practice of the invention into
dosages suitable for systemic administration is within the scope of
the invention. With proper choice of carrier and suitable
manufacturing practice, the compositions of the present invention,
in particular, those formulated as solutions, may be administered
parenterally, such as by intravenous injection. The compounds can
be formulated readily using pharmaceutically acceptable carriers
well known in the art into dosages suitable for oral
administration. Such carriers enable the compounds of the invention
to be formulated as tablets, pills, capsules, liquids, gels,
syrups, slurries, suspensions and the like, for oral ingestion by a
patient to be treated.
[0140] Agents intended to be administered intracellularly may be
administered using techniques well known to those of ordinary skill
in the art. For example, such agents may be encapsulated into
liposomes, then administered as described above. All molecules
present in an aqueous solution at the time of liposome formation
are incorporated into the aqueous interior. The liposomal contents
are both protected from the external micro-environment and, because
liposomes fuse with cell membranes, are efficiently delivered into
the cell cytoplasm. Additionally, due to their hydrophobicity,
small organic molecules may be directly administered
intracellularly.
[0141] Pharmaceutical compositions suitable for use in the present
invention include compositions wherein the active ingredients are
contained in an effective amount to achieve its intended purpose.
Determination of the effective amounts is well within the
capability of those skilled in the art, especially in light of the
detailed disclosure provided herein. In addition to the active
ingredients, these pharmaceutical compositions may contain suitable
pharmaceutically acceptable carriers comprising excipients and
auxiliaries which facilitate processing of the active compounds
into preparations which can be used pharmaceutically. The
preparations formulated for oral administration may be in the form
of tablets, dragees, capsules, or solutions. The pharmaceutical
compositions of the present invention may be manufactured in a
manner that is itself known, e.g., by means of conventional mixing,
dissolving, granulating, dragee-making, levitating, emulsifying,
encapsulating, entrapping, or lyophilizing processes.
[0142] Pharmaceutical formulations for parenteral administration
include aqueous solutions of the active compounds in water-soluble
form. Additionally, suspensions of the active compounds may be
prepared as appropriate oily injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils such as sesame
oil, or synthetic fatty acid esters, such as ethyl oleate or
triglycerides, or liposomes. Aqueous injection suspensions may
contain substances which increase the viscosity of the suspension,
such as sodium carboxymethyl cellulose, sorbitol, or dextran.
Optionally, the suspension may also contain suitable stabilizers or
agents that increase the solubility of the compounds to allow for
the preparation of highly concentrated solutions.
[0143] Pharmaceutical preparations for oral use can be obtained by
combining the active compounds with solid excipient, optionally
grinding a resulting mixture, and processing the mixture of
granules, after adding suitable auxiliaries, if desired, to obtain
tablets or dragee cores. Suitable excipients are, in particular,
fillers such as sugars, including lactose, sucrose, mannitol, or
sorbitol; cellulose preparations such as, for example, maize
starch, wheat starch, rice starch, potato starch, gelatin, gum
tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium
carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If
desired, disintegrating agents may be added, such as the
cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt
thereof such as sodium alginate. Dragee cores are provided with
suitable coatings. For this purpose, concentrated sugar solutions
may be used, which may optionally contain gum arabic, talc,
polyvinyl pyrrolidone, carbopol gel; polyethylene glycol, and/or
titanium dioxide, lacquer solutions, and suitable organic solvents
or solvent mixtures. Dye-stuffs or pigments may be added to the
tablets or dragee coatings for identification or to characterize
different combinations of active compound doses. Such formulations
can be made using methods known in the art (see, for example, U.S.
Pat. Nos. 5,733,888 (injectable compositions); 5,726,181 (poorly
water soluble compounds); 5,707,641 (therapeutically active
proteins or peptides); 5,667,809 (lipophilic agents); 5,576,012
(solubilizing polymeric agents); 5,707,615 (anti-viral
formulations); 5,683,676 (particulate medicaments); 5,654,286
(topical formulations); 5,688,529 (oral suspensions); 5,445,829
(extended release formulations); 5,653,987 (liquid formulations);
5,641,515 (controlled release formulations) and 5,601,845 (spheroid
formulations).
EXAMPLES
[0144] The structure of CCF2/AM used in the experiments described
herein is:
##STR00001##
Example 1
Reduction of Solution Fluorescence
[0145] To investigate the ability of a photon reducing agent to
reduce fluorescence of a solution, emission of a fluorophore from a
sample was monitored in the presence and absence of a photon
reducing agent. The following experiments show that photon reducing
agents can be used to reduce solution based fluorescence from a
fluorophore.
[0146] Fluorescence from a solution of a blue fluorescent dye,
6-chloro-7-hydroxy coumarin 3-carboxylate, was determined in the
presence and absence of a 1 mM phenol red as the photon reducing
agent. A solution of 0.5 .mu.M 6-chloro-7-hydroxy
coumarin-3-carboxylate in solvent containing 50% (vol/vol) aqueous
39 mM phosphate buffer pH 7.5 and 50% (vol/vol) methanol was
prepared. After taking a front face fluorescence spectrum of this
solution on a Spex Fluorolog 2, 1% (vol/vol) of a 100 mM aqueous
Phenol Red stock solution was added and another spectrum taken.
Front face fluorescence refers to exciting the sample at a right
angle to the sample surface and collecting emitted light (for
example, from the excited region or emission region) at 12.5
degrees from such angle of excitation.
[0147] FIG. 2 shows these spectra. Addition of phenol red reduced
front face fluorescence 70 fold. The photon reducing agent, phenol
red, substantially reduced the solution based fluorescence signal.
Although the inventors do not wish to be bound by any proposed
mechanism, at 50% (vol/vol), the organic solvent (methanol) in the
solution may prevent association of the dye molecules in solution.
This is consistent with the explanation that fluorescence reduction
does not require ground-state fluorescence quenching due to dye
stacking. This result is consistent with a photon reducing agent
decreasing fluorescence of the fluorophore by absorbing the
excitation or emission light to or from the fluorophore or by
accepting the energy of the excited singlet state of the
fluorophore (a state that gives rise to the fluorescence) by a
long-range energy transfer mechanism, such as fluorescence
resonance energy transfer.
Example 2
Reduction of Solution Fluorescence is not Necessarily Associated
with Stacking
[0148] To further investigate the ability of a photon reducing
agent to reduce fluorescence of a solution, fluorescence of a dye
in the presence of a photon reducing agent was monitored in the
presence and absence of methanol. The following experiment shows
that a photon reducing agent, phenol red, can be used to reduce
fluorescence from a fluorophore in aqueous solution without dye
stacking.
[0149] The fluorescence from a fluorophore in the presence of a
photon reducing agent (phenol red) was determined in both aqueous
buffer and aqueous buffer containing 50% (vol/vol) methanol. A 10
.mu.M solution of 6-chloro-7-hydroxy coumarin-3-carboxylate was
prepared in 39 mM phosphate buffer pH 7.5 and in a 1:1 mixture of
same phosphate buffer and methanol. 1% (vol/vol) of a 100 mM
aqueous Phenol Red stock solution was added to the solutions.
[0150] FIG. 3 shows front-face fluorescence spectra of these
solutions were obtained on a Spex Fluorolog 2. The presence of
methanol increased fluorescence compared to an all aqueous buffer.
These results further confirm that the fluorescence decrease
observed in Example 1 was not entirely due to dye stacking. Note
the relative fluorescence in Example 2 is comparable or less than
the fluorescence of coumarin/phenol red sample of Example 1.
[0151] The increased fluorescence in the presence of methanol in
the buffer is consistent with finding that 50% methanol buffer
increased 6-chloro-7-hydroxy coumarin-3-carboxylate fluorescence in
the absence of a photon reducing agent by about 30 percent. The
increased fluorescence in the presence of methanol in the buffer is
also consistent with finding that the 50% methanol buffer decreased
the absorbance of the photon reducing agent phenol red at the
emission wavelength of the coumarin by about 20 percent. Under
these conditions, stacking of the dye-based photon reducing agent
and the photon producing agent does not contribute significantly to
fluorescence-reduction effect of dye-based photon reducing agents.
Dye stacking is especially unlikely when the fluorophore is very
water soluble and small, such as 6-chloro-7-hydroxy
coumarin-3-carboxylate.
Example 3
Test of Reduction of Solution Fluorescence Using Non-Dye Quenchers
and Particulates
[0152] To investigate the ability of a candidate photon reducing
agent to reduce fluorescence of a solution, fluorescence of a
fluorophore in the presence of a candidate photon reducing agent
was monitored as a function of photon reducing agent concentration.
The candidate photon reducing agents used were non-dye molecules
and particulates. The following experiments show that non-dye
molecules and particulates can be used as photon reducing agents to
reduce fluorescence from a fluorophore in aqueous buffer.
[0153] Signals from fluorescent dye solutions containing no photon
reducing agents or photon reducing agents, such as Schilling Red
(water, propylene glycol, FD&C Red No. 40 (Allura red),
FD&C Red No. 3 and propyparaben (McCormick & Co., Inc. Hunt
Valley, Md.) and phenol red, were compared to solutions containing
candidate photon reducing agents, non-dye molecules (diatrizoic
acid and Tris (2-amino ethyl) amine) and particulate ink (Higgins
ink). In a 96 well microtiter plate, two fold serial dilutions of
aqueous 0.5 M diatrizoic acid and 1 M tris (2-aminoethyl) amine
(adjusted to pH 7.5 with hydrochloric acid), 100 mM phenol red,
Schilling Red food dye and Higgins ink were prepared in the
presence of 10 .mu.M 6-chloro-7-hydroxy coumarin-3-carboxylate in
the 39 mM phosphate buffer (pH 7.5). The well volume was 100 .mu.l.
A two-fold serial dilution of 10 .mu.M 6-chloro-7-hydroxy
coumarin-3-carboxylate in phosphate buffer pH 7.5 was prepared for
comparison. A linear signal was shown over the range of coumarin
concentrations tested. The fluorescence emission intensity of the
samples was measured on a Cytofluor microtiter plate fluorimeter.
The samples were excited with 395 nm light and fluorescence
emission measured at 460 nm. Data for the Higgins ink and Schilling
Red solutions were normalized by absorbance at 395 nm and 460 nm to
the phenol red solution because their concentrations were either
unknown (Schilling Red) or not defined (Higgins ink).
[0154] FIG. 4 graphs the concentration of the candidate photon
reducing agent against the residual coumarin fluorescence, which
shows the dependence of sample fluorescence on candidate photon
reducing agent concentration. Efficient reduction of fluorescence
occurs at non-dye concentrations greater than 0.5 M, while the
particulate Higgins ink and Schilling Red were similarly effective
to phenol red. This result demonstrates that photon reducing agents
that act as only as collisional quenchers (diatrizoic acid and tris
(2-aminoethyl) amine) will typically require concentrations higher
than 100 mM, which could contribute to ionic strength effects in
potential assays. These results also demonstrate that a photon
reducing agent consisting of light-absorbing (or light scattering)
particulate matter, such as an ink, can effectively reduce solution
fluorescence.
Example 4
Test of Reduction of Solution Fluorescence Using Dye-Based Photon
Reducing Agents with Absorbance Spectra Sufficiently Overlapping
with the Emission or Excitation Spectrum of the Photon Producing
Agent
[0155] To investigate the ability of a candidate dye-based photon
reducing agent to reduce fluorescence of a solution, fluorescence
of a fluorophore in the presence of a candidate photon reducing
agent was monitored as a function of the photon reducing agent
concentration. The candidate photon reducing agents were dye
molecules with different absorption spectra compared to three
different fluorophores. The following experiments demonstrate that
dye based photon reducing agents are most effective in reducing
solution based fluorescence when their absorption maxima
significantly overlaps the excitation and emission spectra of the
fluorophore.
[0156] The efficiency with which water soluble dyes (photon
reducing agents) of different colors were able to reduce
fluorescence from fluorophore solutions of 7-hydroxycoumarin, CCF2,
fluorescein and rhodamine B was studied. A mixture of dye photon
reducing agents with high extinction over the range from 380-555 nm
(named Tararaf) was also studied.
TABLE-US-00001 TABLE 1 Absoption maxima of dyes Naphthol Yellow:
428 (392) nm Tartrazine: 425 nm Phenol Red: 557 (423) nm Acid Red
37: 513 nm Acid Fuchsin: 546 nm Trypan Blue: 607 nm Patent Blue:
635 nm Tararaf: 441 (513) nm (Tararaf contains Tartrazine, Acid Red
37 and Acid Fuchsin in a molar ratio of 5:6:4)
TABLE-US-00002 TABLE 2 Excitation and emission wavelength used in
the study of fluorophores 6-Chloro-7-hydroxycoumarin-3-carboxylate:
ex. 395 nm, em. 460 nm CCF2: ex. 395 nm, em. 530 nm Fluorescein:
ex. 485 nm, em. 530 nm Rhodamine B: ex. 530 nm, cm. 595 nm
[0157] The dye photon reducing agents were made 20 mM in 39 mM
phosphate buffer pH 7.43 containing 10 .mu.M of fluorescent dye.
The Tararaf mixtures concentration was adjusted for its component
Tartrazine to be 20 mM. In a 96-well fluorescence micro titer plate
ten two-fold serial dilutions of these dyes were prepared with
buffer containing 10 .mu.M fluorescent dye. The fluorescence of the
solutions in the wells was measured using a microtiter fluorimeter.
The values were background subtracted and divided by the value
obtained for 10 .mu.M fluorescent dye in the absence of photon
reducing agent. The values so obtained were termed residual
fluorescence.
[0158] FIG. 5 shows 6-chloro-7-hydroxycoumarin-3-carboxylate
solution fluorescence as a function of colored photon reducing
agent concentration. Single yellow dyes that absorb coumarin
excitation and emission light reduced fluorescence at lower
concentrations better than single red or blue dyes. Tararaf also
effectively reduced solution fluorescence. The absorbance spectra
of the components of Tararaf significantly overlap with the
excitation and emission spectra of this fluorophore.
[0159] FIG. 6 shows fluorescein solution fluorescence as a function
of colored photon reducing agent concentration. Single yellow and
red dyes that absorb in the excitation and/or emission spectra of
fluorescein reduced solution fluorescence at lower concentrations
better than blue dyes that absorb predominantly outside that range
of wavelengths. Tararaf also effectively reduced solution
fluorescence. The absorbance spectra of the components of Tararaf
significantly overlap with the excitation and emission spectra of
this fluorophore.
[0160] FIG. 7 shows rhodamine solution fluorescence as a function
of colored photon reducing agent concentration. Red dyes that
absorb in excitation spectrum of rhodamine and blue dyes that
absorb in the emission spectrum of rhodamine reduced solution
fluorescence at lower concentrations more than yellow dyes that
absorb outside that range of wavelengths. Tararaf effectively
reduced solution fluorescence. The absorbance spectra of the
components of Tararaf significantly overlap with the excitation
spectrum of this fluorophore.
[0161] FIG. 8 shows residual CCF2 solution fluorescence as a
function of colored photon reducing agent concentration. Single
yellow and red dyes that absorb in the excitation and/or emission
spectra of CCF2 reduced fluorescence at lower concentrations than
blue dyes that absorb outside that range. Tararaf also effectively
reduced solution fluorescence. The absorbance spectra of the
components of Tararaf significantly overlap with the excitation and
emission spectra of this fluorophore.
[0162] These experiments demonstrate that dye-based photon reducing
agents are most effective in reducing solution based fluorescence
when their absorption maxima lie in the spectral range of the
excitation and/or emission of the fluorophore.
Example 5
Test of Reduction of Solution Fluorescence Using Non-Dye Based
Photon Reducing Agents that Electronically Interact with the Photon
Producing Agent
[0163] To investigate the ability of a candidate transition metal
based or transition metal complex based photon reducing agents to
reduce fluorescence emitted from a solution, fluorescence of a
fluorophore in the presence of a candidate photon reducing agent
was monitored as a function of photon reducing agent concentration.
The candidate photon reducing agents used were ions that can
potentially electronically interact with a fluorophore. The
following experiments demonstrate that non-dye based photon
reducing agents that are transition metal based or transition metal
complexes can be easily tested and selected to reduce solution
based fluorescence of a particular fluorophore. The following
experiments demonstrate that salts of transition metals and their
complexes can act as photon reducing agents of specific
fluorophores.
[0164] Fluorescence was measured with a microtiter plate
fluorimeter with excitation at 395 nm and emission at 460 nm for
the coumarin fluorophore, excitation 485 nm and emission at 530 nm
for fluorescein and excitation 530 nm and emission at 590 nm for
rhodamine B. 500 mM solutions of potassium ferrocyanide (II),
potassium ferricyanide (III), nickel (II) chloride and copper (II)
sulfate were prepared in water. 800 .mu.l of each stock solution
was diluted with 190 .mu.l 150 mM K-MOPS pH 7.15 and 10 .mu.l 1 mM
fluorophore solution to a final 400 mM (stock solution). Two-fold
serial dilutions of these stock solutions into 10 .mu.M fluorophore
containing K-MOPS buffer were prepared in 96 well black (clear
bottom) Costar plates. As in Example 4, the measured values were
background subtracted and normalized to values obtained from
fluorophore in absence of photon reducing agents (see FIG. 9).
[0165] These experiments demonstrated that fluorescence from
coumarin fluorophores can be reduced by iron (III) and nickel (II)
salts in the low millimolar range. Other ions fluorophore
combinations demonstrated an affect at higher concentrations
(approximately 10 mM and above).
Example 6
Test of Reduction of Solution Fluorescence as S Function of Path
Length
[0166] To further investigate the ability of photon reducing agents
to reduce fluorescence emitted from a solution, fluorescence
emitted from a solution containing a fluorophore in the presence of
a photon reducing agent was monitored as a function sample
thickness. The following experiments surprisingly demonstrate that
dye-based photon reducing agents reduce solution fluorescence of a
fluorophore at short transmission distances.
[0167] A photon reducing agent, phenol red, was tested with a
fluorophore, coumarin, as a function of sample thickness (path
length). The experiment was conducted using a microscope equipped
with epi fluorescence. The liquid samples were drawn into low
fluorescence capillary tubes of fixed path length (Vitro Dynamics,
Rockaway N.J.) as indicated in the graphs. The following samples
were evaluated: [0168] 1) 10 .mu.M coumarin (diamonds) [0169] 2) 10
.mu.M coumarin+1 mM phenol red (squares) [0170] 3) 10 .mu.M
coumarin+5% (vol/vol) Schilling Red (triangles).
[0171] The samples were excited using a 405/20 filter via a 425
dichroic reflector through a 20.times. objective (Zeiss 20.times.
Fluar). Emitted light was filtered through a 460/50 filter and
detected by an intensified CCD camera (Stanford Photonics, Menlo
Park, Calif.). The detector output was converted to a 512.times.512
pixel eight-bit digital image. The data reflect the average
intensity within a 20.times.20 pixel area within the capillary. The
background intensity of the field was subtracted from all
values.
[0172] FIG. 10A shows the raw data for this experiment. Coumarin
fluorescence was significantly attenuated by the presence of the
phenol red. Longer paths are also increasingly attenuated.
[0173] FIG. 10B shows the percentage of coumarin fluorescence
observed as a function of path length for each of the dyes tested.
The decrease of fluorescence at short pathlengths is not consistent
with a filtering affect of the photon reducing agent. At short
pathlengths and low concentrations of a photon reducing agent there
is not a sufficient number of photon reducing agent molecules to
filter out light.
[0174] FIG. 10C shows the calculated decrease in coumarin
fluorescence based on filtering affects. A Beer-Lambert
relationship was used to model the expected decrease in
fluorescence due to the effect of filtering light either by
decreasing the amount of light available for excitation of the
fluorophore or the amount of light emitted by the fluorophore.
[0175] These results demonstrate that photon reducing agents can be
effective in reducing solution fluorescence in shallow samples,
such as low volume assay samples. This is a surprising result
because the amount of dye that is located in the space between the
fluorophore and detector in this instance is quite small. This
effect is consistent with deactivation of the excited fluorophore
by fluorescence resonance energy transfer (FRET) to the photon
reducing agents. The average distance between molecules in
millimolar solutions of photon reducing agents is less than 100
.ANG.. At such short distances FRET has been shown to be very
efficient means of quenching fluorophore fluorescence. Although dye
based photon reducing agents can reduce solution fluorescence at
relatively short pathlengths, such photon reducing agents also
allow nearly complete transmission (greater than about 80%) through
shorter path lengths (e.g., less than 15 .mu.m), which is
appropriate for monitoring most mammalian cells.
Example 7
Photon Reducing Agents Reduce Undesired Fluorescence in Cell Based
Assays
[0176] To investigate the ability of photon reducing agents to
reduce undesired fluorescence of a cell-based assay, fluorescence
of a fluorophore in the presence of a photon reducing agent was
monitored using mammalian cells. The following experiments
surprisingly demonstrate that photon reducing agents reduce
solution based fluorescence of a fluorophore in cell-based
assays.
[0177] The fluorescence readout of CCF2 (a substrate for
beta-lactamase) in the presence and in the absence of the photon
reducing agents was measured. The derivative CCF2/AM, as described
in PCT publication WO96/30540 (Tsien), is a vital dye that diffuses
into cells and is trapped by living cells. Cells having esterase
activity that cleaves ester groups on the CCF2/AM molecules, which
results in a negatively charged molecule CCF2 that is trapped
inside the cell. Trapped dye appears as green fluorescence inside
of living cells devoid of beta-lactamase. Cells expressing
beta-lactamase show blue fluorescence because the product of the
beta-lactamase cleavage of CCF2 has blue fluorescence. CCF2 was
incubated with Jurkat cells as previously described (see
WO96/30540). These cells were not attached to the microtiter plates
but are allowed to settle in the plate wells.
[0178] In these experiments, two sets of loading conditions (5 uM
CCF2/AM lot#003 and 10 uM CCF2/AM lot#003) and two types of photon
reducing agents (5% v/v Schilling Red Food Dye and 0.660 mM (final
concentration) phenol red) were used. The presence of photon
reducing agents increased the signal to noise ratio of the assay at
least 200 to 300 percent compared to the absence of a photon
reducing agent. Schilling Red Food Dye can vary from batch to
batch. Thus, it is important to test each batch before using it in
a large series of experiments or cell-based screens. In such
cell-based assays solution fluorescence is typically from a
fluorophore (such as CCF2/AM or its hydrolysis products) in the
cell culture medium that baths the cells.
[0179] Beta-lactamase activity is preferably assessed by addition
of 1/6.sup.th volume CCF2/AM aqueous loading solution containing 6
.mu.M CCF2/AM, 24% PEG-400, 6.2% DMSO, 0.6% Pluronic F127, 7.2 mM
Tartrazine, 7.2 mM Acid Red 40 to microtiter wells at room
temperature. After 30 min incubation, the fluorescence from the
wells is read on a microtiter plate fluorimeter with excitation at
395/20 nm and emission at 460/40 nm and 530/30 nm. The raw
fluorescence emission values were corrected for the signal from
wells devoid of cells. Then, the corrected signal from the blue
channel (460/40 nm) was divided by the signal from the green
channel (530/30 nm). This type of analysis is referred to as
ratioing. With the gain settings used for this experiment, a
population of greater than 95% blue fluorescent cells (>95%
cells expressing beta-lactamase) will give a ratio of greater than
3.0 and a population of entirely green fluorescent cells (no cell
expressing any beta-lactamase) will give a ratio of about
0.1-0.2.
[0180] A comparison to prior art methods to reduce background
fluorescence in cell-based assays was made. In the "washed assay"
format, cells are stimulated to express beta-lactamase, washed,
loaded with CCF2/AM, washed with CCF2/AM free media, and then
plated out into micro-titer plates for fluorescence readout. Such a
protocol with wash steps can work; however, the protocol has
serious limitations and drawbacks for screening, high-throughput
manipulations, and miniaturization. The washed format was compared
with unwashed cells in the absence and presence of a photon
reducing agent (Red Food Dye) at different concentrations ranging
from 0 to 1.064 mM final using a fluorescence readout from a
microtiter plate reader described herein. Photon reducing agents
used in conjunction with the CCF2/AM substrate ester is often
referred to as the "Enhanced Substrate System or ESS."
[0181] FIG. 11 shows that photon reducing agents reduce
fluorescence in unwashed cells and yields signals comparable to
signals from washed cells. Photon reducing agents also provide much
better signals than when no photon reducing agent is present. All
data points are background (buffer plus ESS plus CCF2/AM (no
cells)) subtracted. Data is typically expressed as the ratio of
ratio. The first ratio is the ratio of fluorescence values at the
two indicated emission wavelengths (460 nm/530 nm) for each
experimental data point. The second ratio is the ratio of first
ratio for the two experimental condition of cells constitutively
expressing beta-lactamase and wildtype cells (CMV cells/wildtype
cells).
Example 8
Testing of Dye-Based Photon Reducing Agents for Cytotoxicity in
Cell Based Assays
[0182] To investigate the ability of candidate dye-based photon
reducing agents to reduce undesired fluorescence in a cell based
assay, cytotoxicity in the presence of a candidate photon reducing
agent was monitored as a function of the photon reducing agent
concentration. The candidate photon reducing agents were selected
from a number of dyes based on their absorbance spectra and use
with living systems. The following experiments demonstrate that
dye-based photon reducing agents can be easily tested and selected
for their compatibility with cell-based assays.
[0183] From an initial list of 50 dye compounds, the following dyes
were selected and tested with mammalian cells: Bromophenol Blue,
Chlorophenol Red, Tartrazine, Phenol Red, Naphthol Yellow,
Chromotrope F8, Chromazurol S, Patent Blue, Chromotrope 2R,
Quinoline Yellow, Acid Fuchsin, Erythrosin, Acid Red 37, and
Alizarin Red.
[0184] The toxicity of candidate dye-based photon reducing agents
on mammalian cells was tested with wild-type Jurkat cells. Cells
were incubated in microtiter plate assay wells at room temperature
for 3 hours in the presence of different concentrations of
candidate dye-based photon reducing agents. Propidium iodide was
then added to all wells of the assay plate, and the percentage of
dead cells in each well was estimated. Dead cells did not exclude
propidium iodide.
[0185] FIG. 12 summarizes the results of candidate dye-based photon
reducing agent toxicity testing. Over the concentrations tested
only one candidate dye-based photon reducing agent showed
significant cytotoxicity over a three hour time period. Presumably
at shorter time periods, the candidate dye-based photon reducing
agents will have even less of a cytotoxic effect.
Example 9
Testing of Dye-Based Photon Reducing Agents for Affects on Gene
Activation and Intracellular Enzyme Activity in Cell Based
Assays
[0186] To further investigate the ability of candidate dye-based
photon reducing agents to reduce undesired fluorescence in a
cell-based assay, gene activation and intracellular enzyme activity
in the presence of a candidate photon reducing agent was monitored
as a function of the photon reducing agent concentration. The
candidate photon reducing agents were selected from a number of
dyes based on their absorbance spectra and use with living systems.
The following experiments demonstrate that dye-based photon
reducing agents can be easily tested and selected for their
compatibility with cell-based assays having transcriptional
activity.
[0187] Jurkat cells were treated as described herein for CCF2
experiments. The cells have a G-protein coupled receptor that can
activate a response element controlling the transcription of
beta-lactamase. Cells were stimulated with an agonist for the
G-protein coupled receptor in the presence of different
concentrations of individual candidate dye-based photon reducing
agents that were preincubated with the cells. Cells were then
incubated with CCF2/AM. Cells were subsequently evaluated for
CCF2/AM loading, and conversion to CCF2, and for reporter gene
expression using a microtiter plate fluorimeter. Immediately before
fluorescence readout, the photon reducing agent, Schilling Red Food
Dye, was added to all wells of the assay plate in order to
normalize solution fluorescence. Thus, these experiments were
designed to investigate cell function in the presence candidate
photon reducing agents.
[0188] FIG. 13 illustrates the ability of cells treated with
candidate dye-based photon reducing agents to express
beta-lactamase upon stimulation with agonist, load substrate and
convert substrate. Direct observation of the cells also showed that
the cells loaded substrate to its trapped form, as well as having
beta-lactamase activity. 2-fold dilutions of ESS dyes ranging from
final concentrations of 0.039 mM (left) to 10 mM (right), with the
exception of Patent Blue, which ranged from 0.022 mM (left) to 2.75
mM (right). Data is presented as a ratio of ratios ([agonist
stimulated cells at emission wavelengths 460/530 nm]/[unstimulated
cells emission 460/530 nm]).
[0189] These results demonstrate that cells treated with candidate
dye-based photon reducing agents can load and convert substrate to
its trapped form and support G-protein coupled receptor activation
and reporter gene expression. Substrate loading and trapping
indicates that the cell membrane is intact during candidate photon
reducing agent treatment. Substrate trapping also indicates that
intracellular esterases are sufficiently active to convert CCF2/AM
into its trapped form CCF2. G-protein coupled receptor activation,
gene activation and gene transcription processes also remain active
in the presence of candidate photon reducing agents, as evidenced
by beta-lactamase expression. Finally, beta-lactamase activity is
sufficiently high in cells treated with candidate photon reducing
agents to permit signal detection comparable to signal detection in
the absence of candidate photon reducing agents. In some instances,
signal over background from beta-lactamase expressing cells were
actually increased by the presence of candidate photon reducing
agents, suggesting that combinations of photon reducing reagents
can actually provide superior results.
[0190] These experiments are quite rigorous in the testing of
candidate photon reducing agents because the length of incubation
with such photon reducing agents was approximately three hours and
the photon reducing agents were added prior to gene activation and
expression. In many screening and assay protocols, photon reducing
agents can be added just prior to fluorescence detection, thereby
minimizing the effects such photon reducing agents may have on the
cells or assay.
Example 10
Dye-Based Photon Reducing Agent Sets Reduce Undesired Fluorescence
in Cell Based Assays Better than a Single Dye-Based Photon Reducing
Agent
[0191] To investigate the ability of dye-based photon reducing
agent sets to reduce undesired fluorescence in a cell-based assay,
dye-based photon reducing agent sets were compared with a single
photon reducing agent in the cell-based assays described herein.
Dye-based photon reducing agent sets refer to at least two
dye-based photon reducing agents. The following experiments
demonstrate that dye-based photon reducing agent sets can be yield
better cell based assay results, such as improved signal to noise
ratios and are more robust at protecting against undesired
fluorescence.
[0192] The photon reducing agent considered for use in the sets
were selected from a number of dyes based on the following
criteria: solubility in aqueous solution, having sufficiently high
molar extinction coefficient, having low toxicity to mammalian
cells, and not interfering with gene expression, substrate loading,
and substrate conversion. The following dyes were selected:
Tartrazine, Naphthol Yellow, Chromotrope F8, Chromazurol S, Patent
Blue, Chromotrope 2R, Acid Fuchsin, and Acid Red 37.
[0193] From this list of dyes, two mixtures were created, based on
the absorbance spectra of the dyes. Dyes selection was based on
which dye sets would absorb solution fluorescence over the range of
wavelengths for CCF2 excitation, coumarin emission and fluorescein
emission. The two mixtures were called "ESS Mix 1" and "ESS Mix 2."
ESS Mix 1 was: 100 mM Tartrazine, 100 mM Chromotrope 2R, and 100 mM
Acid Fuchsin. ESS Mix 2 was: 40 mM Tartrazine, 60 mM Acid Red 37,
and 40 mM Acid Fuchsin.
[0194] The ESS dye mixtures were assayed for appropriate
concentrations for optimal use in the homogeneous assay for
beta-lactamase, as described herein. First, dilutions of ESS Mix 1
and ESS Mix 2 were tested using Red Food Dye as a control. In all
cases tested, ESS Mix 1 and ESS Mix 2 improved the fluorescence
readout more than Red Food Dye. Subsequent experiments were used to
evaluate lower concentrations of the ESS mixtures in order to
effectively titrate the amount of the ESS mixtures needed for
optimal assay performance.
[0195] After the initial set of ESS mixture testing, one more
variation of dyes was made. The third ESS mixture was given the
name "Tararaf" (an acronym for Tartrazine, Acid Red 37 and Acid
Fuchsin). Tararaf is: 50 mM Tartrazine, 60 mM Acid Red 37, 40 mM
Acid Fuchsin.
[0196] Tararaf was compared to ESS Mix 1 and ESS Mix 2, as well as
to the individual components of Tararaf and Red Food Dye, in
cell-based assays using CCF2. Tararaf improved the fluorescence
readout more than each of the individual components of the mixture
did.
[0197] FIG. 14 shows the results of these experiments. These
results demonstrate that photon reducing agent sets can improve
signals from cell-based assays compared to either single photon
reducing agents or no photon reducing agents.
PUBLICATIONS
[0198] All publications, including patent documents, world wide web
sites and scientific articles, referred to in this application are
incorporated by reference in their entirety for all purposes to the
same extent as if each individual publication were individually
incorporated by reference.
[0199] All headings are for the convenience of the reader and
should not be used to limit the meaning of the text that follows
the heading, unless so specified.
* * * * *
References